Dilauroylphosphatidylcholine caused a marked increase in progesterone 5 alpha-reductase activity solubilized from rat liver microsomes, whereas naturally occurring phosphatidylcholines from biological sources as well as dioleoylphosphatidylcholine had not effect on the activity. Therefore, the stimulatory effect of phospholipids normally found in rat liver microsomes was examined. The lipid extracts were prepared from the fraction which was freed from 5 alpha-reductase activity by DEAE-cellulose chromatography, and found to exhibit a strong stimulatory effect. The lipid extracts were then separated into phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine by chromatography on silicic acid column and preparative thin-layer plate. Among these endogenous phospholipids, only phosphatidylserine stimulated the 5 alpha-reductase, suggesting that the lipid requirement is specific for phosphatidylserine in steroid 5 alpha-reductase from liver microsomes.