The effects of centrophenoxine, SaH-42348, and DH-990 on several enzymes involved in aminophospholipid biosynthesis in brain have been examined in vitro. Relatively high concentrations of centrophenoxine were required to achieve 50% inhibition of the microsomal enzymes CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase (EPT), CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT), phosphatidyl-N-methylethanolamine N-methyltransferase (PME-NMT), and phosphatidyl-N,N-dimethylethanolamine N-methyltransferase (PDE-NMT). Intermediate concentrations of SaH-42348 inhibited CPT (IC50 = 2.0 mm), EPT (IC50 = 1.9 mm), PME-NMT (IC50 = 0.19 mm), and PDE-NMT (IC50 = 0.17 mm). Of the three drugs tested, DH-990 was the most potent inhibitor of the phospholipid-synthesizing enzymes. Phosphatidylserine decarboxylase, a mitochondrial inner-membrane enzyme [A. K. Percy, J. F. Moore, M. A. Carson, and C. J. Waechter (1983) Arch. Biochem. Biophys.223, 484-494], was virtually unaffected by the three drugs added at millimolar concentrations. Kinetic analyses indicated that the inhibitory action of DH-990 on the brain enzymes was noncompetitive with respect to all substrates. The relatively high sensitivity of CPT (IC50 = 0.6 mm), EPT (IC50 = 2.2 mm), PME-NMT (IC50 = 2.5 μm), and PDE-NMT (IC50 = 2.5 μm) to inhibition by DH-990 in brain microsomes suggests that this compound may be useful for cellular studies on the possible relationships between phospholipid metabolism and neurobiological functions. © 1984.