4-Aminobenzovesamicol was used to test whether activation of protein kinase C protects the vesicular acetylcholine transporter from interaction with vesamicol-like drugs. The essentially irreversible vesamicol analog inhibits the release of newly synthesized [3H]acetylcholine from stimulated hippocampal slices. Prior activation of protein kinase C with a phorbol ester prevented the inhibition of [3H]acetylcholine release, but activation of protein kinase C after the exposure to the irreversible analog did not prevent the effect of the drug. Binding of 4-aminobenzovesamicol in hippocampal synaptosomes, assayed using [3H]vesamicol and back-titration, was decreased by activation of protein kinase C prior to analog exposure but not by activation subsequent to exposure. We propose that phosphorylation of the vesicular acetylcholine transporter prevents the binding of vesamicol-like drugs.