A phospholipase D1 (PLD1) was purified from rat brain by the use of antibody-coupled protein A Sepharose. We found that protein kinase C alp (PKCalpha) stimulated PLD1 activity in the presence of phorbol myristate acetate (PMA). PMA-dependent association of PKCalpha with PLD1 was verified in NIH-3T3 fibroblast cells, and COS7 cells transiently expressing PLD1 as well as in vitro suggesting that the activation of PLD1 resulted from direct association of PKCalpha with PLD1.