The regulation of purified yeast membrane-associated phosphatidylserine synthase (CDP-diacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) and phosphatidylinositol synthase (CDP-diacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) activities by phospholipids was examined using Triton X-100/phospholipid mixed micelles. Phosphatidate, phosphatidylcholine, and phosphatidylinositol stimulated phosphatidylserine synthase activity, whereas cardiolipin and the neutral lipid diacylglycerol inhibited enzyme activity. Phosphatidate was a potent activator of phosphatidylserine synthase activity with an apparent activation constant (0.033 mol %) 88-fold lower than the apparent Km (2.9 mol %) for the surface concentration of CDP-diacylglycerol. Phosphatidate caused an increase in the apparent Vmax and a decrease in the apparent Km for the enzyme with respect to the surface concentration of CDP-diacylglycerol. Phosphatidylcholine and phosphatidylinositol caused an increase in the apparent Vmax for phosphatidylserine synthase with respect to CDP-diacylglycerol with apparent activation constants of 3.4 and 3.2 mol %, respectively. Cardiolipin and diacylglycerol were competitive inhibitors of phosphatidylserine synthase activity with respect to CDP-diacylglycerol. The apparent Ki value for cardiolipin (0.7 mol %) was 4-fold lower than the apparent Km for CDP-diacylglycerol, whereas the apparent Ki for diacylglycerol (7 mol %) was 2.4-fold higher than the apparent Km for CDP-diacylglycerol. Phosphatidylethanolamine and phosphatidylglycerol did not affect phosphatidylserine synthase activity. Phosphatidylinositol synthase activity was not significantly effected by lipids. The role of lipid activators and inhibitors on phosphatidylserine synthase activity is discussed in relation to overall lipid metabolism.