The phospholipases D (PLD1 and 2) are signaling enzymes that catalyze the hydrolysis of phosphatidylcholine to phosphatidic acid, a lipid second messenger involved in cell proliferation, and choline, a precursor of acetylcholine (ACh). In the present study, we investigated development and cognitive function in mice that were deficient for PLD1, or PLD2, or both. We found that PLD-deficient mice had reduced brain growth at 14-27 days post partum when compared to wild-type mice. In adult PLD-deficient mice, cognitive function was impaired in social and object recognition tasks. Using brain microdialysis, we found that wild-type mice responded with a 4-fold increase of hippocampal ACh release upon behavioral stimulation in the open field, while PLD-deficient mice released significantly less ACh. These results may be relevant for cognitive dysfunctions observed in fetal alcohol syndrome and in Alzheimer' disease.