Recently, we have shown that stimulation of [3H]-noradrenaline release from hippocampal slices by 4-aminopyridine (4-AP) is accompanied by an enhancement of the phosphorylation of B-50, a major presynaptic substrate of protein kinase C (PKC). PKC has been implicated in the regulation of transmitter release. In this study, we investigated the effects of 4-AP on B-50 phosphorylation in synaptosomes from rat brain and compared the effects of 4-AP with those of depolarization with K+, in order to gain more insight into the mechanism of action of 4-AP. B-50 phosphorylation was stimulated by incubation with 4-AP for 2 minutes at concentrations ranging from 10 microM to 5 mM. 4-AP (100 microM) stimulated B-50 phosphorylation already within 15 seconds; longer incubations revealed a sustained increase in the presence of 4-AP. B-50 phosphorylation was also stimulated by depolarization with 30 mM K+ for 15 seconds. The effects of both 4-AP or K+ depolarization on B-50 phosphorylation were abolished at low extracellular Ca2+ concentrations. The increase in B-50 phosphorylation induced by 4-AP seemed to be dependent on the state of depolarization, since the effect of 4-AP was largest under nondepolarizing conditions. Comparing the effects of 4-AP and K+ depolarization on B-50 phosphorylation suggests that a different mechanism of action is involved. These results indicate that the stimulation of B-50 phosphorylation by 4-AP in hippocampal slices can be attributed to a direct action of 4-AP on presynaptic terminals. In addition, our results support the hypothesis that B-50 phosphorylation by PKC is involved in Ca2(+)-dependent transmitter release evoked by 4-AP.