In vitro incorporation of [1-14C] acetate into fatty acids and lipid classes of spermatocytes, round spermatids and condensing spermatids enriched by Staput 1 X g sedimentation was measured by thin layer and gas radiochromatography. All three cell fractions showed the full range of de novo synthetase, elongation and desaturase activities necessary for biosynthesis of fatty acids characteristic of mouse testis, but synthesis of fatty acids of greater than 16 carbons declined with progressive stages of differentiation. The magnitudes and patterns of distribution of fatty acid synthesis in the germinal cells were similar to those of whole testis incubated in vitro or injected in vivo with [14C]acetate. On the other hand, complex lipid synthesis was much more variable and incorporation into triacyglycerol was generally much lower in dispersed germinal cells than in whole testis in vitro or in vivo. Cells remained viable throughout the 15-hr incubation. Thus, isolated germinal cells are fully capable of synthesizing their constituent fatty acids, including the long-chain polyenoic acids which they accumulate, but the intratubular environment or association with Sertoli cells may be necessary for maintenance of adequate complex lipid synthesis.