Stimulation of phospholipase D after activation of cell surface receptors has been reported in many cell types. We have investigated the mechanism of activation of this enzyme by collagen in the human platelet by assaying the release of [3H]methylcholine from [3H]methylphosphatidylcholine. Results from these studies suggest that phospholipase D activity is regulated by reversible phosphorylation. Phospholipase D activity was stimulated when platelet-rich plasma was preincubated with collagen and was not inhibited by aspirin. Among various aggregating agents tested, collagen and thrombin but not ADP activated phospholipase D activity (2- to 3-fold). The addition of sphingosine inhibited phospholipase D activity. Preincubation of platelet-rich plasma with sphingosine inhibited collagen- and thrombin-induced platelet aggregation and the release of ATP. The inhibitory effect of sphingosine on collagen- and thrombin- induced platelet aggregation and release of ATP was dose-dependent. The functional significance of phospholipase D activation was also tested by examining the effect of the product, phosphatidic acid, on collagen-induced platelet aggregation and release of ATP. Platelet shape change and the reversibility of platelet aggregation resulted by the addition of phosphatidic acid to platelet-rich plasma. Furthermore, the simultaneous addition of phosphatidic acid and collagen shortened the latency period but had no effect on platelet aggregation. Two platelet proteins (47 kDa and 22 kDa) increased in phosphorylation after the addition of 1 microM phosphatidic acid which did not cause platelet aggregation. These results suggest that collagen stimulates phospholipase D activity which plays a secondary role in platelet aggregation and the release reaction.