The gaseous nature of nitric oxide (NO) has led to the general assumption that its release from neurons during nerve stimulation is independent of vesicular storage. However, recent findings have shown that NO can exist intracellularly as part of more stable bioactive molecules, suggesting that the role of vesicular exocytosis for NO release cannot be excluded simply based on the chemical nature of NO itself. We have used botulinum toxin B (BTX B) to directly address the role of vesicular exocytosis for NO release. BTX B cleaves the synaptic vesicle protein synaptobrevin/VAMP, and by this inhibits Ca++-mediated exocytic release of neurotransmitters. As a target organ we used the guinea-pig enteric nervous system, which innervates the gastrointestinal tract, and in which both classical neurotransmitters as well as NO are released and influence smooth muscle activity. As expected, BTX B (0.1 μm) blocked the nerve stimulation-induced cholinergic and tachykininergic smooth muscle contractions, and markedly inhibited the nerve stimulation-evoked release of [3H]-choline. In contrast, BTX B (0.1 μm) had no effect on nerve stimulation-evoked relaxations, which were equally inhibited by an NO-synthase inhibitor as well as by a selective inhibitor of soluble guanylyl cyclase. In addition, nerve stimulation-evoked NO synthase-dependent outflow of NO/NO2– was unaffected by BTX B (0.1 μm). These findings suggest that the neuronal release of endogenous NO is independent of intact synaptobrevin/VAMP, and therefore provide further evidence that nerve-mediated release of further NO is nonvesicular.