Schizochytrium sp. is a marine microalga that has been developed as a commercial source for docosahexaenoic acid (DHA, C22:6 (omega-3), enriched biomass, and oil. Previous work suggested that the DHA, as well as docosapentaenoic acid (DPA, C22:5 omega-6), that accumulate in Schizochytrium are products of a multi-subunit polyunsaturated fatty acid (PUFA) synthase (1). Here we show data to support this view and also provide information on other aspects of fatty acid synthesis in this organism. Three genes encoding subunits of the PUFA synthase were isolated from genomic DNA and expressed in E. coli along with an essential accessory gene encoding a phosphopantetheinyl transferase (PPTase). The resulting transformants accumulated both DHA and DPA. The ratio of DHA to DPA was approximately the same as that observed in Schizochytrium. Treatment of Schizochytrium cells with certain levels of cerulenin resulted in inhibition of 14C acetate incorporation into short chain fatty acids without affecting labeling of PUFAs, indicating distinct biosynthetic pathways. A single large gene encoding the presumed short chain fatty acid synthase (FAS) was cloned and sequenced. Based on sequence homology and domain organization, the Schizochytrium FAS resembles a fusion of fungal FAS beta and alpha subunits.