1. The responses to non-adrenergic, non-cholinergic nerve stimulation have been compared with those to exogenously applied ATP on seventeen different tissues from a number of vertebrate classes.2. Stimulation of all the mammalian gut preparations studied (with the exception of the guinea-pig ileum) after blockade of the effects of adrenergic and cholinergic nerve stimulation by guanethidine (3.5 muM) and hyoscine (1.3 muM) caused inhibition; exogenously applied ATP mimicked this inhibitory response.3. Stimulation of the guinea-pig ileum in the presence of hyoscine and guanethidine, usually caused a diphasic response, relaxation followed by contraction; exogenously applied ATP mimicked this response, in contrast to acetylcholine and noradrenaline which caused excitation and relaxation respectively.4. Stimulation of preparations of lower vertebrate gut and guinea-pig bladder in the presence of hyoscine and guanethidine caused contraction; exogenously applied ATP mimicked this contractile response.5. In each preparation the time course of the response to ATP was similar or identical to the response to non-adrenergic, non-cholinergic nerve stimulation.6. The results are consistent with the hypothesis that a purine nucleotide may be the transmitter substance released from non-adrenergic, non-cholinergic nerves supplying smooth muscle preparations from a number of vertebrate classes.