In studies of isometric contractions in prostatic portions of rat vas deferens evoked by single pulse electrical stimulation, the K+ channel blockers 4-aminopyridine, tetraethylammonium and charybdotoxin, but not apamin, significantly reduced the prejunctional inhibitory potency and the maximum inhibitory effect of the alpha 2-adrenoceptor agonist xylazine. The protein kinase C activator phorbol dibutyrate had similar effects to 4-aminopyridine against xylazine. However, 4-aminopyridine, tetraethylammonium, charybdotoxin and phorbol dibutyrate, but not apamin, significantly increased the magnitude of the isometric contraction to a single stimulus. 4-Aminopyridine and phorbol dibutyrate significantly reduced, while tetraethylammonium did not affect, isometric contractions to noradrenaline, and 4-aminopyridine failed to affect contractions to alpha,beta-methylene-ATP, so that the effects of these agents on the isometric contraction to a single stimulus were presumably by a prejunctional action. The Ca2+ entry facilitator Bay K 8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyrid ine carboxylic acid methylester) increased stimulation-evoked contractions by a postjunctional action and reduced the inhibitory effects of xylazine. When the isometric contraction following 4-aminopyridine was reduced by decreasing the stimulation voltage or by reducing the Ca2+ concentration from 2.5 to 0.9 mM, 4-aminopyridine significantly reduced the potency of xylazine. However, tetraethylammonium and Bay K 8644 failed to affect the inhibitory potency of xylazine in low Ca2+. It is concluded that the K+ channel blocker 4-aminopyridine reduces the prejunctional inhibitory potency of xylazine, and this action is independent of increased neurotransmitter release. These results suggest that prejunctional alpha 2-adrenoceptor-mediated inhibition in rat vas deferens involves K+ channels sensitive to block by 4-aminopyridine.