A major goal of this work was to determine in NIH 3T3 fibroblasts whether the recently described effects of H2O2 on phospholipase D-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) are mediated by similar or different mechanisms. While exposure of NIH 3T3 fibroblasts to H2O2 stimulated the hydrolysis of both PtdEtn and PtdCho, the following important differences were noted: (i) prolonged (24 h) treatment of fibroblasts with 400 nM phorbol 12-myristate 13-acetate (PMA) blocked the stimulatory effect of H2O2 on PtdEtn, but not on PtdCho, hydrolysis; (ii) PMA-induced hydrolysis of PtdEtn, but not PtdCho, was inhibited by H2O2; (iii) the stimulatory effect of H2O2 was additive with that of sphingosine or staurosporine, inhibitors of protein kinase C, on the hydrolysis of PtdCho, but not PtdEtn; (iv) with membranes isolated from H2O2-treated fibroblasts, the hydrolysis of PtdCho, but not PtdEtn, was increased compared to values obtained with control membranes. These results imply that H2O2 regulates PtdEtn and PtdCho hydrolysis by different mechanisms. Stimulation of PtdEtn hydrolysis by H2O2, sphingosine, and staurosporine may commonly involve, at least in part, neutralization of an inhibitory protein kinase C isozyme.