Phosphorothioate analogues of adenosine 5'-triphosphate (ATP) have been tested on the rat and guinea-pig vas deferens, the guinea-pig taenia coli and urinary bladder. Adenosine 5'0-(2-thiotriphosphate) (ATP beta S) was more active than adenosine 5'0(1-thiotriphosphate) (ATP alpha S) and ATP in producing contractile responses on the vas deferens of rat and guinea-pig, and guinea-pig bladder, though the difference of potency was less marked for producing relaxation of the carbachol-contracted taenia coli. No differences were observed between the A and B diastereoisomers of ATP alpha S or ATP beta S. Contractions of the vas deferens produced by ATP alpha S were of much longer duration than those produced by ATP beta S. When tested against electrically-evoked twitch responses of the vas deferens the order of potencies was reversed with ATP being most active and ATP beta S least active. These inhibitory effects were blocked by 8-phenyl-theophylline. The calculated pA2 values for ATP, adenosine, beta, gamma-methylene ATP (APPCP) and ATP alpha S were similar, suggesting a common site of action. The results do not reveal any stereoselectivity among the tissues tested, for the diastereoisomers of ATP phosphorothioates; the observed differences of potency may be due to differences between ATP alpha S and ATP beta S in their rates of metabolism to adenosine. The different response profiles to the phosphorothioates may however reflect some differences of receptor mechanisms.