In our continuing search for insecticidal substances from microbial metabolites, we recently found two active compounds from Penicillium. They showed the marked insecticidal activity toward the 3rd instar larvae of silkworm upon oral administration. One of them, okaramine A (1),1>3) is a new type of indole alkaloid with a seven-ring system, the other one, okaramine B (2),2'3) being a derivative with a eitht-ring system. Since we were interested in the unique structure of okaramines, we tried to determine if okaramines or similar compounds were produced by other strains. As a result, Penicillium simplicissimum AHU8402 showed activity almost equivalent to AK-40, the producing strain of okaramines.4) In this paper, we report the isolation and structural elucidation of the insecticidal constituents of AHU8402. From an acetone extract of mycelia and media of this strain, three insecticidal compounds were isolated. Two of them were identified as okaramine A (1) and okaramine B (2), and the third one seemed to be a new congener, which was termed okaramine C (3). High-resolution El mass spectrometry of 3 established its molecular formula as C32H36N4O3. The 1H-NMR(Table I) and 13C-NMR(Table II) spectra showed the presence of 3a-hydroxy-A^8-(a,a-dimethylallyl)-l,2,3, 3a,8,8a-hexahydropyrrolo[2,3-6]indole-2-carboxylic acid and a 2,3-disubstituted indole with a diketopiperazine ring, indicating that only the azocine ring part of 1 was changed in 3. A comparison between the molecular formulae of 1 and 3 proved 3 to be a tetrahydro derivative of 1. The !H-NMR and proton-proton coupling correlation (COSY) spectra indicated that 3 had additional vinyl (<5 4.97, d, 7= 10.6 Hz; 3 5.05, d, 7=17.6Hz; and 3 6.16, dd, 7=17.6, 10.6Hz) and -CH2-CHC (S 3.04, dd, 7=15.2, ll.0Hz; 3 3.64, dd. 7=15.2, 4.3Hz; and 3 4.38, dd, 7=11.0, 4.3Hz) groups, strongly suggesting that the CY=C2' double bond was saturated and that the N3'-C4' bond was reductively cleaved. This presumption was supported by the fact that 3 lacked the UV absorption at 374nm that was present in 1, and by the fact that 3 had another exchangeable proton at 2.88ppm in the ^-NMR spectrum. These data led us to formulate the structure of 3 as that shown in Fig. 1. The proposed structure is also supported by the 13C-NMR data (Table II). Signals of a methine carbon at 115.9ppm assigned to CT and of a quaternary carbon at 148.9ppm assigned to C2' in 1 were observed, respectively, at 26.7ppm as a methylene carbon and at 57.0ppm as a methine carbon in 3. Furthermore, signals of a methine carbon at 123.6ppm assigned to C5' and of a methine carbon at 139.8ppm assigned to C4' in 1 were observed, respectively, at 147.6ppm as a methine carbon and at 112.5ppm as a methylene carbon in 3. The relative configuration of the pyrrolidine ring in 3 is considered to be the same as that in 1, because protons at C2 and C3 in 3 had chemical shifts and coupling constants quite similar to those of 1. Therefore, the planar structure of okaramine C (3) is concluded to be the structure shown in Fig. 1. The relative configuration at C2' remains uncertain. Okaramines exhibit insecticidal activity toward the larvae of silkworms upon oral administration. The LD50 values of okaramine A (1) and okaramine B (2) were 8 and 0.2/ig/g of diet, respectively. The LD50 value of okaramine C (3), whose azocine ring is cleaved, was 7/ig/g of diet, suggesting that the azocine ring moiety does not play an essential role in exhibiting the activity.