The effect of activation of protein kinase C by phorbol esters has been studied on the nonmyelinated (C) fibers of the rat vagus nerve. Grease-gap recording at room temperature was used to monitor changes in the resting and action potentials. Effects of phorbol esters on the rate of efflux of 86Rb and 14C-guanidinium were also measured. The active isomer beta-phorbol 12,13-dibutyrate (PDBu), applied for 10 min at concentrations of 10 nM to 3 microM, caused a slowly developing depolarization, which persisted after the drug was washed out. The action potential was concomitantly reduced. These effects did not occur with the inactive isomer alpha-phorbol 12,13-didecanoate. The PDBu-induced depolarization was reduced by about 75% if Na+ was replaced by the impermeant cation N-methyl-(+)-glucamine (NMG); the residual effect was almost abolished if the nerves were presoaked in a solution containing gluconate in place of Cl-. It was concluded that increases in conductance mainly to Na+ and Cl- were responsible for the depolarization. The response was unaffected by tetrodotoxin or calcium-channel blockers. Omission of Ca2+, surprisingly, enhanced the PDBu-induced depolarization 3-5-fold; furthermore, addition of 2 mM Ca2+ following a PDBu-induced depolarization recorded in Ca2+-free solution caused a pronounced repolarization. This effect of Ca2+ occurred also with Sr2+ and Ba2+, but not with other divalent cations or with La3+. Divalent cations known to block Ca channels inhibited the repolarizing action of Ca2+. These results suggested that Ca2+ acts intracellularly, either to block Na channels opened by PDBu or to activate protein phosphatases. The PDBu-induced response in Ca2+-free solution was increased 2-fold by a reduction in pH from 7.4 to 6.5. Under normal conditions the nerve was reversibly depolarized by this pH change; after PDBu this pH sensitivity was enhanced, and depolarization occurred at a less acidic pH. PDBu caused a 3-4-fold increase in the rate of efflux of 86Rb (a marker for K+ ions) and of 14C-guanidinium (a marker for Na+ ions) from preloaded nerves. These effects, in contrast to the depolarization, were transient.(ABSTRACT TRUNCATED AT 400 WORDS)