The structure-activity relationships for a variety of adenine nucleotide analogues at P(2x)- and P(2Y)-purinoceptors were investigated. Compounds formed by structural modifications of the ATP molecule including substitutions of the purine ring (C2, C8, N1, and N(6)-substituents, and a uridine base instead of adenine), the ribose moiety (2' and 3'-positions), and the triphosphate group (lower phosphates, bridging oxygen substitution, and cyclization) were prepared. Pharmacological activity at P(2Y)-purinoceptors was assayed in the guinea pig taenia coli, endothelial cells of the rabbit aorta, smooth muscle of the rabbit mesenteric artery, and turkey erythrocyte membranes. Activity at P(2X)-purinoceptors was assayed in the rabbit saphenous artery and the guinea-pig vas deferens and urinary bladder. Some of the analogues displayed selectivity, or even specificity, for either the P(2X)- or the P(2Y)-purinoceptors. Certain analogues displayed selectivity or specificity within the P(2X)- or P(2Y)-purinoceptor superfamilies, giving hints about possible subclasses. For example, 8-(6-aminohexylamino)ATP and 2',3'-isopropylidene-AMP were selective for endothelial Pzypurinoceptors over P(2Y)-purinoceptors in the guinea pig taenia coli, rabbit aorta, and turkey erythrocytes. These compounds were both inactive at P(2X)-purinoceptors. The potent agonist N(6)-methyl ATP and the somewhat less potent agonist 2'-deoxy-ATP were selective for P(2Y)-purinoceptors in the guinea pig taenia coli, but were inactive at P(2X)-purinoceptors and the vascular P(2Y)-purinoceptors. 3'-Benzylamino-3'-deoxyATP was very potent at the P(2X)-purinoceptors in the guinea pig vas deferens and bladder, but not in the rabbit saphenous artery and was inactive at P(2Y) receptors. These data suggest that specific compounds can be developed that can be utilized to activate putative subtypes of the P(2X)- and P(2Y)-purinoceptor classes.