The function of protein kinase C (PKC) is closely regulated by its subcellular localization. We expressed PKCalpha fused to green fluorescent protein (PKCalpha-GFP) and examined its translocation in living and permeabilized cells of the human parotid cell line, HSY-EB. ATP induced an oscillatory translocation of PKCalpha-GFP to and from the plasma membrane that paralleled the appearance of repetitive Ca2+ spikes. Staurosporine attenuated the relocation of PKCalpha-GFP to the cytosol and caused a stepwise accumulation of PKCalpha-GFP at the plasma membrane during ATP stimulation. Diacylglycerol enhanced the amplitude and duration of the ATP-induced oscillatory translocation of PKCalpha-GFP. Ionomycin induced a transient translocation of PKCalpha-GFP to the plasma membrane despite the continuous elevation of cytosolic Ca2+. The ionomycin-induced transient translocation of PKCalpha-GFP was prolonged by staurosporine, diacylglycerol, and phorbol myristate acetate. Experiments using permeabilized cells showed that staurosporine or the elimination of ATP and Mg2+ decreases the rate of dissociation of PKCalpha-GFP from the membrane. Diacylglycerol slowed the dissociation of PKCalpha-GFP from the membrane regardless of the Ca2+ concentration. The effect of diacylglycerol was attenuated by ATP plus Mg2+ at low concentrations of Ca2+ (<500 nm) but not at high concentrations of Ca2+ (>1000 nm). These data suggest a complex interplay between Ca2+, diacylglycerol, and phosphorylation in the regulation of the membrane binding of PKCalpha.