The induction of mammalian cell proliferation requires the expression of a specific set of genes. Tumor promoters stimulate cell growth by activating the Ca*+ and phospholipid-dependent protein kinase, protein kinase C (PKC). DNA topoisomerase I, a nuclear enzyme involved in transcription, was phosphorylated by activated PKC in vitro. Phosphorylation by PKC stimulated the DNA relaxation activity of topoisomerase I two-to threefold. Therefore, DNA topoisomerase I is a substrate for PKC-mediated activation by phosphorylation and may serve as a nuclear target of mitogenic signals generated by tumor promoters in vivo.