Two γ-glutamylpeptides, γ-L-glutamyl-L-2-aminohex-4-ynoic acid and γ-L-glutamyl-L-erythro-2-amino-3-hydroxyhex-4-ynoic acid, were isolated from fruit bodies of Tricholomopsis rutilans. Identifications were based on elementary analysis, hydrolysis, IR spectra, optical rotation and analysis of dansyl derivatives. Previously we reported the characterization of L-2-aminohex-4-ynoic acid [1], L-2-amino-3-hydroxyhex-4-ynoic acid (threo and erythro) [2,3] and L-3-(4-carboxy-3-furyl)alanine [4,5] from fruit bodies of Tricholomopsis rutilans (Fr.) Sing. Column chromatography on Dowex 1 revealed that the fungus also contains two unknown ninhydrin-positive substances (A and B). Both compounds A and B gave the normal violet coloration with ninhydrin. Mild hydrolysis of A with N H₂SO₄ produced L-2-aminohex-4-ynoic acid and L-glutamic acid (molar ratio, 1:1.16), which were separated on a Dowex 1-column and identified by TLC, IR, elementary analysis and optical rotation. Hydrolysis of dansyl A yielded dansylglutamic acid. Compound B was hydrolysed with N H₂SO₄ to give L-erythro-2-amino-3-hydroxyhex-4-ynoic acid and L-glutamic acid (molar ratio, 1:0.85). Hydrolysis products and its dansyl derivative were separated and identified as before. The results of elementary analysis supported the structures, γ-L-glutamyl-L-2-aminohex-4-ynoic acid and γ-L-glutamyl-L-erythro-2-amino-3-hydroxyhex-4-ynoic acid. It is worth noting that the threo-isomer could not be detected in the hydrolysates of the relevant fractions of compound B, even before the crystals of B separated. The threo-isomer, if present, would be expected to be eluted from the Dowex 1-column in the same fractions as the erythro-form. The free amino acids of threo- and erythro-form occur in this fungus in the ratio 2:3 [3]. During investigations of the constituents of the fruits of Piper officinarum, we have reported previously the presence of 3 new compounds namely, Me piperate [1], N-isobutyl-trideca-13-(3,4-methylenedioxyphenyl)-2,14,12-trienamide [2] and N-isobutyl docosa-trans-2-trans-4-cis-10-trienamide [3] (filliline). We now wish to report the structure of another new isobutylamide from the same source. The petrol extract of the fruits, on repeated column chromatography over neutral Al₂O₃ and repeated recrystallization, gave a white waxy crystalline compound mp 67-67.5°. The compound analysed for C₂₄H₄₃NO (found C, 79.82; H, 12.34; N, 3.61; calc. for C₂₄H₄₃NO: C, 80.2; H, 12.08 and N, 3.59%). The UV spectrum (MeOH) λ 259 nm, indicated the presence of a conjugated system related to sorbamide [4,5]. IR (KBr) showed characteristic bands for -NH₂ (3300, 3080 cm⁻¹), -C=O (1625 cm⁻¹), -C=C₁ (1660 cm⁻¹), -C=C₂ (1620 cm⁻¹), -(CH=CH)₂ (997 cm⁻¹) and no peak in the region 960-965 cm⁻¹; indicating the presence of a trans-2-trans-4-dienamide system [4,5]. The 100 MHz PMR (CDCl₃) spectrum showed a doublet at 0.9 δ (9H, J=6 Hz) which has been assigned to -CH(CH₃)₂ and terminal -CH₃ protons, a broad singlet at 1.28 δ due to methylene groups (18H), a multiplet between 1.8 and 2.22 δ (6H) attributed to allylic protons, a triplet centred at 3.13 δ accounting for two protons (N-CH₂-C-), typical for isobutylamides, which is replaced by a doublet after D₂O exchange. The PMR also showed a triplet at 5.33 δ due to two cis protons (-CH=CH-), a doublet at 5.78 δ (J=16 Hz) attributed to one α-olefinic proton adjacent to a carbonyl group, a multiplet between 5.65 to 6.24 δ accounting for two (γ, δ) olefinic protons and a broad multiplet at 7.2 δ attributed to one (β) olefinic proton. Because of the conjugated nature of the carbonyl with two double bonds, as shown by the IR and UV spectra of the compound, it appears that one of the double bonds (isolated) is located elsewhere in the chain. The appearance of a triplet at 5.33 δ (2H) in the PMR spectrum indicated the presence of identical olefinic protons of an isolated double bond and were thus assigned as cis. The KMnO₄ oxidation of the compound to dodecanoic acid showed the exact location of this cis isolated double bond in the molecule. MS of the compound with fragments at m/e 361 (M⁺), 333, 289, 261, 254, 236, 223, 208, 192, 180, 156, 152, 121, 115, 96, 95 and 81 supported its structure as the isobutylamide of eicosa-trans-2-trans-4-cis-8-trienamide.