The role of protein kinase C (PKC) in platelet-activating-factor (PAF)-induced platelet activation was examined by using two selective inhibitors of PKC, namely Ro 31-7549/001 and Ro 31-8220/002. Both inhibitors dose-dependently inhibited PAF-induced phosphorylation of the major 40-47 kDa protein substrate of PKC, with 50% inhibition at 4.5 microM-Ro 31-7549/001 and 0.7 microM-Ro 31-8220/002. Inhibition of PKC had no effect on maximal elevation of intracellular Ca2+ [Ca2+]i produced by either a high or a low dose of PAF, but significantly increased the duration of the Ca2+ signal and the thromboxane B2 (TxB2) generation in high-dose PAF-stimulated platelets. The inhibitors also abrogated the effect of the PKC activator phorbol 12-myristate 13-acetate on PAF-induced [Ca2+]i elevation. Sub-maximal PAF-induced dense-granule release and platelet aggregation were dose-dependently inhibited by Ro 31-7549/001 and Ro 31-8220/002. The findings suggest that endogenously activated PKC holds a bifurcating role in PAF-activated platelets, negatively affecting duration of both [Ca2+]i and TxB2 generation, and positively influencing dense-granule release and aggregation.