1. The effects of alpha,beta-methylene ATP (alpha,beta-MeATP) on membrane properties and excitatory junction potentials (EJPs) were examined in smooth muscle cells of the guinea-pig, rabbit and pig bladder. 2. Intracellular recording with microelectrodes was used to record membrane electrical activity from the guinea-pig bladder. ATP (10(-3) M) produced a rapid, large depolarization with a marked increase in spike frequency, while carbachol (10(-4) M) or acetylcholine (ACh; 10(-4) M) produced only a small or no depolarization with a smaller increase in spike frequency. alpha,beta-MeATP produced a similar response to that of ATP but at a much lower concentration (5 x 10(-6) M), and the response was transient even in the continuous presence of this agent. 3. Changes in the membrane potential and conductance elicited by alpha,beta-MeATP were also measured with the double sucrose-gap method. alpha,beta-MeATP (5 x 10(-6) M) depolarized the membrane and increased the membrane conductance in all three species, but both parameters returned to control values during continuous exposure to this agent. 4. Intracellular recording with microelectrodes showed that in the guinea-pig bladder treatment with alpha,beta-MeATP abolished the response to ATP, while the response to ACh was unchanged. 5. With the double sucrose-gap method, EJPs were elicited by transmural nerve stimulation of strips of the guinea-pig, rabbit and pig bladder and had spikes superimposed, leading to contractions. Desensitization of P2-purinoceptors by alpha,beta-MeATP (3-5 x 10(-6) M) abolished the EJPs and spikes, and reduced the contraction. Atropine (10(-6) M) alone did not alter the EJPs but reduced the contraction. Combined application of both agents abolished the contraction. 6. It is concluded that in the guinea-pig, rabbit and pig bladder ATP is an excitatory transmitter with ACh and EJPs are mediated by ATP.