Progression of human prostate cancer to a malignancy that is refractory to androgen-ablation therapy renders the disease resistant to available treatment options and accounts for the high prostate cancer mortality rate. Epidermal growth factor receptor (EGFR) expression in human prostate cancer specimens increases with disease progression to androgen-refractory prostate cancer, and experimental models implicate EGFR-dependent signaling to Erk1/2 activation in the androgen-refractory prostate cancer phenotype. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced Erk1/2 activation in human prostate cancer PC-3 cells is a paradigm of diacylglycerol-induced EGFR transactivation in androgen-independent prostate cancer. In this report, we establish an obligatory role for TPA-induced protein kinase C (PKC)-α activation in EGFR transactivation and signaling to Erk1/2 activation in PC-3 cells. TPA-regulated molecules include PKCs, PKDs, and Ras guanyl nucleotide-releasing proteins. The PKC-selective inhibitors GF109203X and Gö6983 each blocked TPA-induced EGFR transactivation, indicating a requirement for PKC. PC-3 cells express four PKC isozymes. Prolonged bryostatin 1 treatment abrogated PKCα expression without altering expression levels of the other PKC isozymes. Pharmacologic PKCα “knockdown” abrogated TPA-induced Erk1/2 activation without affecting the EGF/EGFR-induced response, indicating that PKCα was required for EGFR transactivation but dispensable for signaling of ligand-activated EGFR to Erk1/2 activation. We corroborated this by showing that Gö6976, which is a PKCα-selective inhibitor in PC-3 cells, likewise abolished TPA-induced Erk1/2 activation and did not inhibit EGF/EGFR-induced Erk1/2 activation. Gö6976 had similar effects in DU145 cells, providing evidence for a common PKCα-dependent Erk1/2 activation mechanism in androgen-independent human prostate cancer cells of distinct genetic origin. These results constitute a rational basis for selective PKCα inhibition as a modality of prostate cancer therapy.