In order to determine if ATP contributes as an excitatory transmitter in the guinea-pig bladder, experiments were conducted with ANAPP3, a photoaffinity analogue of ATP, which is an antagonist of adenine nucleotides in several other smooth muscles. With or without photoactivation with visible light, ANAPP3 antagonized contractile responses of in vitro strips of bladder to exogenous ATP. The antagonism was specific in that responses to acetylcholine and KCl were not affected by ANAPP3. Responses of strips of bladder to transmural electrical stimulation were not antagonized by ANAPP3 and were relatively insensitive to atropine. However, combined treatment with ANAPP3 and atropine produced a marked antagonism of the neurogenic response. In experiments with bladders obtained from animals pretreated with 6-hydroxydopamine, the ANAPP3-sensitive component of the neurogenic response was absent. These results suggest that acetylcholine, released from cholinergic nerves, and a purine, released from 6-hydroxy-dopamine-sensitive nerves, are both involved in motor transmission in this tissue.