Despite their widespread use in investigations of protein kinase C (PKC), concern is often expressed regarding the specificity of action of phorbol esters. We have extensively compared the effects of PDBu, a phorbol ester that activates PKC, with those of its inactive analog, 4 alpha-PDBu, on calcium (Ca) channel regulation in acutely isolated guinea pig hippocampal neurons and found that PKC-dependent and - independent actions could be clearly distinguished. While both phorbol esters depressed whole-cell barium current through Ca channels (IBa), PDBu was approximately 100-fold more potent than 4 alpha-PDBu. PKC- independent effects began to appear in the range of 5–10 microM, doses that, while high, have been used in some investigations. Moreover, only PDBu (1) was active when applied intracellularly, (2) had effects that were blocked by the PKC inhibitor H-7, and (3) induced PKC translocation with potency similar to its potency in depressing IBa. The finding that 4 alpha-PDBu acted only extracellularly was unexpected and suggested either that it acted via an extracellular binding site or that its orientation in the membrane was crucial to its effects on Ca channels. Finally, (4) PDBu alone caused a hyperpolarizing shift in the voltage dependence of the high-voltage-activated, rapidly inactivating (N type) component of Ca current. This result extends our previous finding that the N-type current component was depressed by PDBu to a greater extent than the L-type component and may represent an important new mode of neurotransmitter regulation of ion channels in the brain via PKC.