The fungus Pithomyces chartarum produces the cyclohexadepsipeptide sporidesmolide III (molecular formula C₂₃H₃₆N₄O₈), which shows infrared absorptions for ester carbonyl (5.72μ), amide I (5.95, 6.09μ), and amide II (6.45, 6.55μ) groups. Acid hydrolysis yields valine (~1 mol), leucine (~2 mol), and α-hydroxy-β-methylbutyric acid (~2 mol). By applying the previously established mass spectral rationale for cyclohexadepsipeptides (sporidesmolides I and II) and combining with hydrolysis results, 16 possible structures were excluded via mass spectral analysis—including active hydrogen determination using N-deuteriated sporidesmolide III (showing four active hydrogens)—leading to the deduction of a unique structure (C) where the N-methyl group of sporidesmolide I is replaced by N-H. The α-hydroxy-β-methylbutyric acid from hydrolysis was identified as the L-isomer. Sporidesmolide III consumed ~2 equivalents of sodium hydroxide to produce acids identical to the known sporidesmolic acid A; carboxypeptidase digestion further confirmed the amino acid sequence, and the asymmetric center configuration sequence was determined to be L,D,D,L,L,L. Thus, sporidesmolide III is N-desmethyl-sporidesmolide I.