The regulation of phosphatidylcholine-hydrolyzing phospholipase D (PLD) by protein kinase C (PRC) in membranes of Chinese hamster lung fibroblasts (CCL39) was studied using conventional PKC isoforms alpha, beta and gamma isolated from rat brain and recombinant PKC isoforms. Cells were incubated with [14C]choline to label endogenous phosphatidylcholine before membranes were prepared and assayed for release of [14C]choline. PKC alpha was the most potent activator of PLD, producing a maximal effect at approximately 0.1 microgram/ml. PKC beta also stimulated PLD but was less potent and less efficacious, whereas PKC gamma was ineffective. Stimulation required addition of a PKC activator, but the isoform specificity was the same whether phorbol 12-myristate 13-acetate (PMA) or Ca2+ was used. Recombinant Ca(2+)-independent PKC isoforms delta, epsilon, and zeta failed to stimulate PLD, but recombinant PKC beta 1 stimulated PLD in a manner similar to the purified brain PKC beta. Immunoblot analysis of the soluble fraction of CCL 39 fibroblasts detected only the alpha and zeta isoforms of PKC. The results suggest that PKC alpha and beta are activators of PLD and that PKC alpha is responsible for the activation in these fibroblasts.