We found that extracellular ATP can increase the intracellular Ca2+ concentration ([Ca2+]i) in mouse pineal gland tumor (PGT-beta) cells. Studies of the [Ca2+]i rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5'-O-(3-thiotriphosphate) > or = UTP > 2-chloro-ATP > 3'-O-(4-benzoyl)benzoyl ATP, GTP > or = 2-methylthio ATP, adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) > CTP. AMP, adenosine, alpha,beta-methyleneadenosine 5'-triphosphate, beta,gamma-methyleneadenosine 5'-triphosphate, and UMP had little or no effect on the [Ca2+]i rise. Raising the extracellular Mg2+ concentration to 10 mM decreases the ATP- and UTP-induced [Ca2+]i rise, because the responses depend on the ATP4- and UTP4- concentrations, respectively. The P2U purinoceptor-selective agonist UTP and the P2Y purinoceptor-selective agonist ADP beta S induce inositol 1,4,5-trisphosphate generation in a concentration-dependent manner with maximal effective concentrations of approximately 100 microM. In sequential stimulation, UTP and ADP beta S do not interfere with each other in raising the [Ca2+]i. Costimulation with UTP and ADP beta S results in additive inositol 1,4,5-trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45-55%, whereas it has no effect on the ADP beta S response. Treatment with 1 microM phorbol 12-myristate 13-acetate inhibits the ADP beta S-induced [Ca2+]i rise more effectively than the ATP- and UTP-induced responses. These results suggest that P2U and P2Y purinoceptors coexist on PGT-beta cells and that both receptors are linked to phospholipase C.