Rat testicular 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-Isomerase) converts pregnenolone to progesterone. The enzyme is localized to the microsomal membranes of testicular homogenates, and treatment of the microsomes with phospholipases causes a reduction in 3 beta-HSD-Isomerase activity. The relationship between the membrane microenvironment and 3 beta-HSD-Isomerase activity was investigated by adding phospholipids of known structure to microsomal incubations and determining the effects on the conversion of pregnenolone to progesterone. Phosphatidylcholines with saturated acyl chains of 8, 10, 12, and 14 carbon atoms, or unsaturated chains, were extremely inhibitory to 3 beta-HSD-Isomerase, causing reductions in specific activity to 10-40% of the control value. Furthermore, the inhibition appeared to be caused primarily by a reduction in the active enzyme concentration (Vmaxapp). Phosphatidylcholines with longer saturated acyl chains were without effect. Phosphatidylserine and phosphatidic acid were activators of 3 beta-HSD-Isomerase. These phospholipids decreased the Kmapp value (to 21% and 43% of control values, respectively), suggesting that the enhancement of 3 beta-HSD-Isomerase activity was through an active-site-oriented effect. Furthermore, for phosphatidic acid to activate 3 beta-HSD-Isomerase, saturated acyl chains of 16 carbon atoms were necessary, other configurations being slightly inhibitory. The remarkable specificity for certain phospholipid configurations and the different effects of these membrane components suggest that androgen biosynthesis may be regulated by changes in the phospholipid microenvironment of 3 beta-HSD-Isomerase.