ABSTRACTA membrane‐associated CDP‐1,2‐diacyl‐sn‐glycerol (CDP‐diacylglycerol):myo‐insitol phosphatidyltransferase activity (phosphatidylinositol synthase, EC 2.7.8), was identified and partially characterized from a microsome preparation of germinating soybean seeds. The pH optimum for enzyme activity was pH 8.2 with Tris‐HCl buffer. The dialyzed enzyme preparation had an absolute requirement for manganese (5 mM) or magnesium ions (50 mM). Increasing levels of Triton X‐100 at molar ratios of 1:1 to 12:1 of Triton X‐100 to CDP‐diacylglycerol stimulated phosphatidylinositol synthase activity (about 10‐fold) to a maximum followed by an inhibition of activity as the molar ratio was raised beyond the point of maximum activity. Phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol,sn‐glycero‐3‐P,and choline inhibited activity while nucleotides stimulated activity. Phosphatidylinositol synthase activity was found to be thermally inactivat