Vasoactive intestinal peptide release and L-[3H]citrulline production were examined in ganglia isolated from the myenteric plexus of guinea-pig intestine. The nicotinic agonist, 1,1-dimethyl-4-phenylpiperizinium stimulated vasoactive intestinal peptide release and L-[3H]citrulline production; the latter was considered an index of nitric oxide production. Both vasoactive intestinal peptide release and L-[3H]citrulline production were abolished by tetrodotoxin, hexamethonium, and the nitric oxide synthase inhibitor, NG-nitro-L-arginine. Inhibition of vasoactive intestinal peptide release by NG-nitro-L-arginine was reversed by L-arginine but not by D-arginine. Exogenous nitric oxide stimulated vasoactive intestinal peptide release whereas exogenous vasoactive intestinal peptide had no effect on L-[3H]citrulline production. The pattern of stimulation by nitric oxide and inhibition by NG-nitro-L-arginine implied that vasoactive intestinal peptide release is facilitated by and may be dependent on nitric oxide production. Consistent with this notion, vasoactive intestinal peptide release in response to either 1,1-dimethyl-4-phenylpiperizinium or nitric oxide was abolished by KT 5823, an inhibitor of cyclic GMP-dependent protein kinase activity and by LY83583, an inhibitor of soluble guanylate cyclase activity. The study provides the first direct evidence of nitric oxide production from enteric ganglia.