Cholinephosphotransferase activity in brain microsomes may be assayed with labeled alkylacylglycerols or with CDP-choline with label in the phosphocholine with nearly identical results. The direct linear plot method was used for evaluation of Michaelis-Menten kinetic parameters. Most of the cholinephosphotransferase activity is in microsomes and a stimulatory factor seems to be present in the cytosol. Incubation of microsomes with labeled alkylacylglycerols and CDP-choline, in the initial absence of CDP-ethanolamine, produced labeled ethanolamine glycerophospholipids as well as labeled choline glycerophospholipids. Since the labeling of ethanolamine glycerophospholipids was increased by the addition of CMP, the labeling was probably due to the reversal of ethanolamine phosphotransferase to yield CDP-ethanolamine produced by the choline phosphotransferase reaction. Cholinephosphotransferase was reversed more readily than ethanolaminephosphotransferase in brain as it is in liver (Kanok and Ohno, 1973). Only trace quantities of plasmalogens were formed with labeled alkylacylglycerols. Previous results of plasmalogen labeling from labeled CDP-nucleotides were apparently due to reversal of phosphotransferase reactions. Alkylacylglycerophospholipids are not good substrates for plasmalogen formation, even when they are incorporated into microsomes.