Atrial natriuretic factor (ANF) suppresses adrenergic and purinergic neurotransmission in the rabbit vas deferens. The neuromodulatory mechanism of action for ANF is not established, but is thought to be independent of cyclic GMP (cGMP) generation. This study directly tested for an involvement of ANF R1 receptors, the receptors coupled to guanylate cyclase activation, in neuromodulatory effects of ANF. The experiments utilized the novel ANF R1 receptor antagonists, anantin and A71915. ANF inhibited both purinergic and adrenergic neurotransmission in a concentration-dependent manner. Neither anantin nor A71915 altered basal neurotransmission or neuromodulatory responses to ANF. Both antagonists significantly attenuated cGMP generation in response to ANF. In contrast, the suppression of cyclic AMP concentrations caused by ANF was sustained in the presence of A71915 but not anantin. The results are consistent with prior observations dissociating neuromodulatory effects of ANF from an elevation in cGMP concentrations. Additionally, ANF increased cGMP concentrations in denervated preparations, suggesting that most if not all ANF R1 receptors are in non-neuronal tissue in the vas deferens. The novel aspect of this study involves the use of selective ANF R1 receptor antagonists to test directly for a neuromodulatory role of ANF R1 receptors. The inability of these structurally distinct ANF R1 receptor antagonists to prevent the neuromodulatory effects of ANF suggests the involvement of ANF R2 (clearance) receptors or another unidentified ANF receptor in mediating the response.