The biochemical mechanisms underlying the process of facilitation in sympathetic nerve terminals of the guinea-pig vas deferens have been investigated by intracellular and focal extracellular recording techniques. Activation of protein kinase C by the phorbol ester, phorbol 12,13-dibutyrate (PDBu) greatly increased the amplitude of excitatory junction potentials (e.j.ps) from the first stimulus in a train and altered the pattern of facilitation. The configuration of the extracellularly recorded nerve terminal impulse and the sensitivity of the postjunctional membrane to released ATP were unaffected. The specific protein kinase C inhibitor, Ro-31,8220, abolished facilitation and antagonized the effects of PDBu. These results suggest that protein kinase C plays a fundamental role in ATP release from sympathetic nerves and in particular in the mechanisms underlying facilitation.