The fatty acid composition of the total, neutral, sterol, free fatty acid, and polar-lipid fractions in the mycelium of Choanephora cucurbitarum was determined. The major fatty acids in all lipid fractions were palmitic, oleic, linoleic, and γ-linolenic acid. Different lipid fractions did not show any particular preference for any individual fatty acid; however, the degree of unsaturation was different in different lipid fractions. Free fatty acid and polar lipid fractions contained a higher proportion of γ-linolenic acid than did triglyceride and sterol fractions. Addition of glutamic acid to the malt–yeast extract medium resulted in the biosynthesis of a number of long-chain fatty acids beyond the γ-linolenic acid. These fatty acids, e.g., C22:1, C24:0, and C26:0, were never observed to be present in the fungus when grown on a malt–yeast extract medium without glutamic acid. Furthermore, thin-layer chromatographic analysis showed a larger and denser spot of diphosphatidyl glycerol from the mycelium grown on glutamic acid medium than from the control mycelium. The possible significance of this finding is discussed.