The structures of cytochalasin K, L and M, isolated from the fungus Chalara microspora, have been determined by spectroscopic methods, primarily ¹H NMR and ¹³C NMR. In an investigation of toxic metabolites produced by the fungus Chalara microspora (Corda) Hughes, three new cytochalasins have been isolated. Previously, the methyl ester of (+)-3,4 anhydroshikimic acid (1)¹, chaloxone (2)²˒³ and chalmicrine (3)⁴ were isolated from the same fungus. The fungus was grown in a stationary culture⁴, and both medium and mycelium were extracted with EtOAc. The cytochalasins were isolated using reversed phase chromatography. Cytochalasin K (4) was amorphous, [α]ᵈ⁵ -177°(EtOH); mol. formula C₃₂H₃₇NO₆ (High res. MS: 531.2660; calcd. 531.2621); UV (abs. ethanol): 232 nm (11300); IR (KBr): 3380 (OH), 1750, 1700 (broad) (C=O). 270 MHz ¹H NMR (Fig. 1), including decoupling experiments, established chemical shifts and coupling constants for nearly all protons. A comparison of these data with those of chaetoglobosin A (1) and its acetate⁵, indicated that cytochalasin K is identical with the acetate of chaetoglobosin A, except that the indolyl group of the latter is replaced by a phenyl group. ¹³C NMR of cytochalasin K (Fig. 1) is also consistent with the proposed structure A⁶. Cytochalasin L (5) was also amorphous, [α]ᴰ -165°(EtOH); mol. formula C₃₂H₃₇NO₇ (High res. MS: 547.2535; calcd. 547.2570); UV (abs. ethanol): no characteristic absorption maxima were discerned; IR (KBr): 3400 (OH), 1720 (broad) (C=O). ¹H NMR and ¹³C NMR of cytochalasin L (Fig. 2) were compared with those of cytochalasin K (A), and resulted in structure 5 for cytochalasin L. The ¹³C NMR shift for C₉ increases due to substitution with oxygen, while the shift for C₂₃ decreases on going from a ketone to an ester. Cytochalasin M (6) crystallized from MeOH/H₂O, mp. 161-162°C, [α]ᵈ +18.7°(EtOH); mol. formula C₃₀H₃₇NO₆ (High res. MS: 507.2708; calcd. 507.2631); UV (abs. ethanol): 235 nm (11300); IR (KBr): 3380 (OH), 1750, 1705 (broad) (C=O), 1670 (C=C). ¹H NMR and ¹³C NMR of cytochalasin M (Fig. 3) revealed the disappearance of the fragment -OOC-CH=CH-CO-CHOAc- compared to cytochalasin L (5), but the existence of -OOC-, two -CH₂- groups, -CHOH- and an α,β-unsaturated carbonyl group. These features, with the ester replaced by a ketone, are found in chaetoglobosin F (8)⁷, and as a consequence cytochalasin M was assigned structure 6. The hydroxyl group was placed in the 20-position, as in chaetoglobosin F (8), to comply with the oxygenation pattern found in cytochalasin K (4) and L (5), and in the chaetoglobosins. The structure for cytochalasin M (6) was verified by an X-ray analysis⁸. Biogenetically, cytochalasin L (5) may be derived from cytochalasin K (4) by a Baeyer-Villiger type reaction, for which there is precedence among other cytochalasins⁹.