Adenine nucleotides inhibited isoproterenol- and forskolin-stimulated cyclic AMP accumulation in C6-2B rat glioma cells. Inhibition occurred in the presence of a phosphodiesterase inhibitor, and no effect of adenine nucleotides was observed in direct measurements of phosphodiesterase activity in intact cells. Pretreatment of C6-2B glioma cells with pertussis toxin blocked the inhibitory effects of P2Y-purinergic receptor agonists. The pharmacological specificity for a series of ATP and ADP analogs (2-methylthioadenosine 5'-triphosphate > or = 2-methylthioadenosine 5'-diphosphate > adenosine 5'-O-(2-thiodiphosphate) > 2-chloro-adenosine 5'-triphosphate = ADP = adenosine 5'-O-(3-thiotriphosphate) > ATP > UTP) was similar to that expected of a P2Y-purinergic receptor; the P2X-purinergic receptor agonists, alpha,beta-methyleneadenosine 5'-triphosphate and beta,gamma-methylene-adenosine 5'-triphosphate, had no effect. Because activation of phospholipase C occurs in response to P2-purinergic receptor activation in many target tissues, the effects of P2Y-receptor agonists on inositol phosphate accumulation were measured in C6-2B cells. No evidence for P2Y-purinergic receptor-mediated regulation of inositol lipid metabolism was observed under conditions where muscarinic cholinergic receptor activation or AIF4-markedly increased inositol phosphate accumulation. These results suggest that a P2-purinergic receptor subtype with distinct signaling properties exists on C6-2B rat glioma cells. Although this receptor expresses the general pharmacological specificity of a phospholipase C-coupled P2Y-purinergic receptor, it may represent a unique receptor subtype since it inhibits adenylyl cyclase.