The subcellular distribution and activation state of protein kinase C (PKC) was studied after short-term exposure of rabbit platelets to platelet-activating factor (PAF). Cytosolic and nonidet P-40-solubilized particulate extracts prepared from treated platelets were subjected to analytical column chromatography on MonoQ, hydroxylapatite and Superose 6/12. PKC activity was assayed by the ability of the enzyme to phosphorylate the following substrates: (i) histone H1 in the presence of the activators calcium, diacylglycerol and phosphatidylserine; (ii) histone H1 following proteolytic activation of PKC with 0.5 micrograms trypsin/ml; and (iii) protamine in the absence of calcium and lipid. PAF treatment for 1-20 min elicited a rapid 2-4-fold activation of both cytosolic and particulate-derived PKC as assessed by all three methods. On the other hand, there were no significant PAF-induced changes in the level of [3H]phorbol-12,13-dibutyrate binding by soluble and particulate-associated PKC. Hydroxyapatite column chromatography revealed that in non-treated rabbit platelets the type II (beta) form of PKC predominated, but PAF appeared to induce a shift in the elution profile from this resin. The stability of the PAF activation of PKC to column chromatography and the altered binding affinity to hydroxylapatite indicated that the stimulation might be a consequence of covalent modification, albeit minor, since PKC still eluted as an 80 kDa protein from Superose 6/12. As the PAF-induced increases in the kinase activity of PKC were preserved even after proteolytic activation with trypsin, but were without effect on the phorbol ester binding activity, such a putative modification may have occurred within or near the catalytic domain of PKC. These findings imply that PAF may directly modulate the activity of preexisting membrane-associated PKC by a novel mechanism, rather than by eliciting its recruitment from the cytoplasm.