In neuroblastoma x glioma hybrid NG108-15 cells, ATP induced a concentration-dependent increase in the intracellular Ca2+ concentration ([Ca2+]i), accompanied by inositol phosphate formation. Under the same conditions, we found a marked increase in cAMP levels produced by ATP at concentrations similar to those required to increase [Ca2+]i. The Ca2+ ionophore A23187 or bradykinin, which evoked inositol phosphate formation and increases in [Ca2+]i, did not increase, and instead slightly decreased, cAMP content, indicating that ATP-induced cAMP accumulation was not due to activation of Ca(2+)-sensitive adenylyl cyclase. The effect of ATP on cAMP production was not dependent on generation of adenosine caused by ATP hydrolysis. Among several P2 purinoceptor agonists, adenosine-5'-O-(3-thio)triphosphate, 5'-adenylylimidodiphosphate, and adenosine-5'-O-(2-thio)diphosphate evoked both cAMP accumulation and Ca2+ mobilization. In contrast, beta,gamma-methylene-ATP selectively elicited cAMP accumulation, whereas 2-methylthio-ATP and UTP induced only Ca2+ mobilization, without affecting cAMP levels. The potent P2x purinoceptor agonist alpha,beta-methylene-ATP did not induce cAMP accumulation or Ca2+ mobilization. The cAMP accumulation induced by ATP was not affected by the P2 receptor antagonist suramin but was inhibited by P1 receptor antagonists such as 8-(p-sulfophenyl)theophylline, 3-isobutyl-1-methylxanthine, and xanthine amine congener. However, the ATP-induced increase in [Ca2+]i was not affected by suramin or xanthine amine congener. Taken together, these results indicate that ATP activates two distinct purinoceptors that are coupled to different signal transduction systems, one being adenylyl cyclase and the other phospholipase C, in NG108-15 cells. Furthermore, pharmacological profiles of the adenylyl cyclase-coupled receptor were quite different from those of any known purinoceptor subtypes, especially in the unusual sensitivity of the receptor to P1 and P2 receptor agonists and antagonists. It is therefore suggested that ATP-induced cAMP accumulation may be mediated by a novel subtype of purinoceptor in NG108-15 cells.