Supplementation with choline during pregnancy in rats causes a long-lasting improvement of visuospatial memory of the offspring, The biochemical mechanism of this effect may be related to the function of choline as a precursor of phosphatidylcholine (PC), the substrate of a receptor-stimulated enzyme, phospholipase D (PLD), PLD activation initiates the sequential formation of two intracellular messengers, phosphatidic acid and 1,2-sn-diacylglycerol, We hypothesized that prenatal choline status may cause long-term modulation of PLD-catalyzed PC hydrolysis in the hippocampus, a brain region implicated in visuospatial memory functions, PLD activity was determined in hippocampal slices prelabeled with [H-3]glycerol or [H-3]oleic acid by measuring the PLD-catalyzed formation of [3H]phosphatidylpropanol in the presence of l-propanol, Slices were obtained from male pups born to mothers consuming a control diet, a choline-supplemented diet, or a choline-free diet from days 11 to 17 of pregnancy, The radiolabeling of phospholipid classes was unaffected by the treatments, Prenatal choline supplementation significantly increased basal PLD activity in [H-3]glycerol-labeled slices [by 46% of controls on postnatal day (P) 7 and by 36% on P21], and [H-3] oleate-labeled slices (by 91% on P7), as well as glutamate-stimulated PLD activity in [H-3]oleate-labeled slices (by 60% on P?), Prenatal choline deficiency failed to alter PLD activity, The actions of choline apparently required intact cells because in vitro assays of PLD activity in hippocampal homogenates, using fluorescent NBD-PC as substrate, revealed no differences between groups, The results show that prenatal choline supplementation up-regulates basal and receptor-stimulated PLD activity in the hippocampus during postnatal development.