Phospholipase D (PLD) in lymphocytic mouse leukemic L1210 cells has been found to be activated by oleate both in vitro and in intact cells. The PLD activity was measured by phosphatidylethanol produced from radiolabeled phosphatidylcholine or myristic acid in the presence of ethanol. This oleate-activated PLD was further characterized in intact cells and compared with that in HL60 cells. Unlike PLD in HL60 cells, the PLD in L1210 cells was activated by unsaturated fatty acids, stimulated by melittin, insensitive to guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), ADP-ribosylation factor (ARF) and phosphatidylinositol 4,5-bisphosphate (PIP2), independent of phorbol 12-myristate 13-acetate (PMA) and staurosporine, and inhibited by pervanadate. These observations indicate that the PLD present in L1210 cells is distinct from that in HL60 cells. Key PLD properties of L1210 cells such as insensitivity to GTP gamma S, ARF, PIP2, or PMA were in good agreement with currently known in vitro properties of the oleate-activated PLD found in mammalian sources. Therefore, the L1210 cells could be used as an intact-cell source for an oleate-activated PLD.