The phorbol esters in addition to being among the most potent mouse skin tumour promoters profoundly affect many different biological systems. It is postulated that they act through activation of protein kinase C, but substantial heterogeneity in their pharmacological and binding behaviour in some systems has caused concern about whether this is their only target. Evidence linking protein kinase C activation with biological responses to the phorbol esters includes similarity in structure-activity relations for binding and response; in vitro phosphorylation of specific proteins by protein kinase C at the same sites at which phorbol ester treatment induces phosphorylation in intact cells; and correlation in certain cell types between down regulation of protein kinase C on chronic phorbol ester treatment and loss of cellular responsiveness to the phorbol ester. Here we report that microinjection of purified protein kinase C into Swiss 3T3 fibroblasts pretreated with the phorbol ester phorbol 12,13-dibutyrate (PDBu) restores the mitogenic response of the cells to PDBu, directly establishing the involvement of protein kinase C in this response.