Protein kinase C is the Ca{dollar}\sp{lcub}2+{rcub}{dollar}/phospholipid-dependent enzyme that serves as the receptor for, and is directly activated by, the tumour-promoting phorbol esters. To examine the involvement of protein kinase C in the regulation of the organization or function of the actin-containing microfilaments, the activity of the enzyme towards two distinct groups of proteins that are thought to be involved in microfilament regulation has been investigated. For these studies, protein kinase C was partially purified from bovine brain or was more extensively purified from rat brain. Two proteins that are localized in certain areas of microfilament-membrane attachment (focal contacts), vinculin and talin, were identified as in vitro substrates for protein kinase C. Purified protein kinase C also phosphorylated chicken gizzard myosin light chain kinase and different forms of caldesmon, proteins that are involved in the regulation of contractile events. Chicken gizzard caldesmon and chicken liver caldesmon{dollar}\sb{lcub}72{rcub}{dollar} as well as two forms of bovine live caldesmon (caldesmon{dollar}\sb{lcub}150{rcub}{dollar} and caldesmon{dollar}\sb{lcub}77{rcub}{dollar}) were all in vitro substrates for protein kinase C. The sites of phosphorylation of the substrate proteins were examined by phosphopeptide mapping and phosphoamino acid analysis. Phosphorylation of chicken gizzard caldesmon by protein kinase C partially abolished its inhibitory activity towards the actin-activated ATPase of skeletal muscle myosin and diminished associations between caldesmon and actin.;Treatment of intact human platelets with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a tumour-promoter that activates protein kinase C in living cells, stimulated phosphorylation of talin and caldesmon{dollar}\sb{lcub}77{rcub}{dollar}. The phosphate content of talin was elevated by 44%, but the apparent stoichiometry of phosphorylation was low. In contrast, the phosphate content of caldesmon{dollar}\sb{lcub}77{rcub}{dollar} increased approximately 4-fold. Moreover, the phosphopeptides that appeared in response to TPA treatment had the same migration pattern as the two major phosphopeptides of bovine liver caldesmon{dollar}\sb{lcub}77{rcub}{dollar} phosphorylated in vitro.;The results of this study imply that protein kinase C, by phosphorylating focal contact proteins or proteins involved in the control of contractile events, may have a role in microfilament regulation