Extracellular ATP (10(-3) M) stimulated [3H]phosphatidylcholine secretion approximately 3.4-fold in rat type II pneumocytes prelabeled overnight with [3H]choline. The same concentration of ATP caused a rapid increase in [3H]inositol trisphosphate (IP3) and a decrease in [3H]phosphatidylinositol bisphosphate (PIP2) in [3H]inositol-prelabeled cells. ATP also caused a biphasic increase in 1,2-[3H]diacylglycerol in cells prelabeled with [3H]arachidonic acid: a rapid increase that peaked at 10 s followed by a larger increase that peaked at 5-10 min. The first peak in diacylglycerol and the increase in IP3 are consistent with phospholipase C action on PIP2 and generation of second messengers that promote mobilization of intracellular Ca2+ and activation of protein kinase C. However, at the level of phosphatidylcholine secretion the stimulatory effects of ATP and of direct activators of protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1,2-dioctanoyl-sn-glycerol, were at least additive, suggesting that activation of protein kinase C may not be the major signal transduction mechanism in ATP action or alternatively that ATP activates a different isoform of protein kinase C. Pretreatment of type II cells with TPA for 30 min led to a subsequent 40% diminution in the stimulatory effects of ATP on both phosphatidylcholine secretion and IP3 generation.