The present study provides evidence that rat brain protein kinase C elicits a phosphotransferase activity towards histone and undergoes autophosphorylation in the absence of phosphatidylserine. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate binds to and activates protein kinase C in a phospholipid-free reaction. The apparent activation constant (Ka = 2.7 nM) is not modified by the absence of phospholipid but the maximum velocity is greatly decreased. The phosphotransfer reaction to exogenous substrates occurs in 0.5 mM ethylene-bis(oxyethylenenitrilo)tetraacetic acid, although autophosphorylation in these conditions requires the presence of Ca2+. The protein kinase C inhibitor (1-(5-isoquinolinesulfonyl)-2-methylpiperazine inhibits the reaction, whereas the cAMP-dependent protein kinase inhibitor is ineffective. In contrast to diacylglycerol, which is a poor activator, unsaturated fatty acids potently activate the phospholipid-free reaction. Moreover, the substrate specificity is markedly changed, e.g., myelin basic protein and histone types VI-S and VII-S appear to be relatively better substrates in the phospholipid-free reaction. The data presented indicate that protein kinase C (or some individual isoforms) may function, at least partially, without binding to membrane phospholipid and suggest that this novel characteristic of phorbol esters may account for their tumor-promoting activity.