Acetylcholine (ACh) was found to markedly enhance the nerve stimulation induced twitch response of isolated, field-stimulated rat vas deferens (RVD). The ED200 (concentration which enhances the twitch response to 200% of control) for this potentiation was 6 X 10(-6)M with the maximum twitch response being increased by more than 3 fold (325 +/- 30%). Carbachol (ED200 = 8.5 X 10(-7)M) showed identical results. With each drug the potentiation was competitively antagonized by atropine (10(-7) - 10(-5)M). Physostigmine 10(-8) - 10(-6)M) both enhanced the basal twitch response (215 +/- 8% of control at 10(-5)M) and the sensitivity of the RVD to ACh (ED200 = 3.3 X 10(-7)M) but not to carbachol. Atropine, on the other hand reduced the basal twitch response by 18 +/- 3% at 10(-5)M. Hemicholinium (10(-4)M) also reduced the basal twitch responses by 23 +/- 5%. ACh (10(-7)M - 10(-5)M) did not modify the responses of unstimulated RVD to norepinephrine or KCl suggesting a pre-synaptic site of action. Taken together these results are compatible with the presence of a pre-junctional, excitatory muscarinic mechanism in the field stimulated RVD. That this cholinergic system may be of physiological significance is supported by the observations that atropine and hemicholinium depress while physostigmine enhances the twitch response in the absence of exogenous ACh.