Nitric oxide (NO) inhibits the release of acetylcholine and cholinergic contractions in the small intestine of several species, but no information is available about the mouse ileum. This study examines the effects of NO on the electrically evoked release of [3H]acetylcholine and smooth muscle contraction in myenteric plexus-longitudinal muscle preparations of wild-type mice and of neuronal NO synthase (nNOS) and endothelial NOS (eNOS) knockout mice. The NOS inhibitor N(G)-nitro-L-arginine (L-NNA) and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) concentration dependently increased the evoked [3H]acetylcholine release and cholinergic contractions in preparations from wild-type mice and from eNOS knockout mice. Effects of L-NNA were specifically antagonized by L-arginine. In contrast, L-NNA and ODQ did not modify the release and contractions in preparations from nNOS knockout mice. The NO donor S-nitroso-N-acetyl-DL-penicillamine inhibited the electrically evoked release of [3H]acetylcholine and longitudinal muscle contractions in a quantitatively similar manner in wild-type preparations as well as in nNOS and eNOS knockout preparations. We conclude that endogenous NO released by electrical field stimulation tonically inhibits the release of acetylcholine. Furthermore, data suggest that nNOS and not eNOS is the enzymatic source of NO-mediating inhibition of cholinergic neurotransmission in mouse ileum.