As lead has been shown to activate protein kinase C (PKC), and gliomas are reported to be highly dependent on PKC for their proliferation, this study was undertaken to investigate whether lead may act as a mitogen in human astrocytoma cells, and to determine the role of PKC in this effect. Lead acetate (from 100 nM to 100 microM) induced a concentration- and time-dependent increase in DNA synthesis, as measured by incorporation of [methyl-3H]thymidine into cell DNA, without causing any cytotoxicity. Flow cytometric analysis showed that lead was able to stimulate the cell cycle transition from the G0/G1 phase to the S/G2 phase, resulting in increased percentage of cells in the latter phase. Western blot analyses showed that lead induced translocation of PKCalpha, but not of PKCepsilon or PKCzeta, from the cytosolic to the particulate fraction, with a concomitant increase in PKC enzyme activity. Prolonged exposure to lead caused down-regulation of PKCalpha, but not of PKCepsilon. The effect of lead on DNA synthesis was mediated through PKC as evidenced by the finding that two PKC inhibitors, GF 109203X and staurosporine, as well as down-regulation of PKC through prolonged treatment with 12-O-tetradecanoylphorbol 13-acetate, blocked lead-induced DNA synthesis. Further experiments using a pseudosubstrate peptide targeting classical PKCs and selective down-regulation of specific PKC isoforms indicated that the effect of lead on DNA synthesis was mediated by PKCalpha. Altogether, these results suggest that lead stimulates DNA synthesis in human astrocytoma cells by a mechanism that involves activation of PKCalpha.