ATP (EC50 5 microM) induced endothelium-dependent relaxation of the isolated aorta of the newborn pig, but the other naturally occurring nucleotides CTP, GTP, ITP and UTP were more than 100 times less potent. 2-Methylthio-ATP (EC50 0.1 microM) was 50 times more potent than ATP, but the unnatural enantiomers L-ATP and 2-methylthio-L-ATP were virtually inactive. beta,gamma-Imido-ATP and beta,gamma-methylene-ATP, both of which are resistant to degradation by ectonucleotidases on cultured pig endothelial cells, were much less potent than ATP. ADP beta S, which is also resistant to degradation, was equipotent with ATP at low concentrations but achieved a maximal relaxation of only 50% that of ATP. The Rp and Sp diastereoisomers of ATP beta S were both equipotent with ATP at low concentrations and both achieved approximately 60% of the maximal relaxation of ATP. The Rp and Sp diastereoisomers of ADP alpha S were both less potent than ATP and achieved only approximately 25% of the maximal relaxation of ATP. These results demonstrate that the P2-purinoceptor mediating endothelium-dependent relaxation of the pig aorta exhibits a high degree of specificity for the adenine base, is stereospecific for the D-ribofuranosyl moiety, requires a phosphate chain of 2 or 3 units but is not stereoselective toward this phosphate chain. These structural requirements have some features in common with the P2-purinoceptors on smooth muscle and on platelets, and are quite different from those of the ectonucleotidases present on pig endothelial cells in culture.