The twitch response observed during low frequency electrical stimulation of postganglionic cholinergic neurones supplying the longitudinal smooth muscle of the guinea-pig ileum was markedly reduced by incubation with an homogeneous preparation of botulinum type A neurotoxin (4.3-8.6 nM). This intoxication of the autonomic cholinergic neurones was long-lasting, irreversible by washing, but readily reversed by 4-aminopyridine (50-1000 microM). The noradrenergic motor response of the rat anococcygeus following field stimulation was partially antagonised by the neurotoxin. The non-adrenergic inhibitory response of the guinea-pig taenia coli, elicited by field stimulation, was not antagonised by botulinum toxin, suggesting that a source of a non-adrenergic inhibitory transmitter exists, other than intramural cholinergic neurones. However, the neurogenic excitatory responses of the guinea-pig bladder, elicited by field stimulation in the presence of atropine and guanethidine, were virtually abolished by botulinum toxin. It is suggested that the parasympathetic neurones which supply the smooth muscle of the guinea-pig urinary bladder co-release acetylcholine and a non-cholinergic excitatory transmitter; ATP or polypeptides are possible candidates.