Conventional protein kinase C (cPKC) isoforms are activated by a coincident rise in cytosolic Ca(2+) and membrane-bound diacylglycerol. In excitable cells, cPKC may be activated by Ca(2+) influx through voltage-gated Ca(2+) channels (VGCC). cPKCs, in turn, are known to modulate the activity of VGCC. We examined whether PKCalpha, a cPKC, could be activated by depolarization in a neuroendocrine cell line and whether activation occurred on a time scale that modulated the depolarization-evoked intracellular Ca(2+) concentration ([Ca(2+)](i)) signal. Pheochromocytoma cells (PC12 cells) were transfected with wild-type and mutant forms of PKCalpha labeled with yellow fluorescent protein to monitor kinase translocation. Simultaneously, [Ca(2+)](i) changes were monitored with fura-2. Two point mutations that render PKCalpha inactive, D187A in the Ca(2+) binding site and K368R in the ATP binding site, significantly prolonged the time-to-peak of the depolarization-evoked [Ca(2+)](i) signal. A mutation that modulates membrane insertion (W58G) and two mutations of an autophosphorylation site (S657A, S657E) had no effect on the kinetics of the [Ca(2+)](i) signal. We conclude that in PC12 cells, Ca(2+) entry through VGCC rapidly activates PKCalpha, and that PKCalpha can modulate the Ca(2+) signal on a physiologically relevant time scale. Point mutations of PKCalpha can be used as specific and potent modulators of the PKC signaling pathway.