Adenine and uridine nucleotide-promoted inositol phosphate accumulation was studied in HEK293 cells. Concentration effect curves for ADP, ATP, and 2ClATP were complex and could be resolved by a two-site model into low and high potency components, suggesting the involvement of two receptors. The maximal effect observed for the P2Y1 receptor-selective agonists 2MeSATP and 2MeSADP was 65-70% of that observed with ATP, ADP, or 2ClATP, and the concentration effect curves for these two analogs were consistent with their interaction at a single site. The P2Y1 receptor-selective antagonist PPADS completely blocked 2MeSATP-stimulated inositol phosphate accumulation, but only partially antagonized the response to ATP. UTP also was an agonist, but the maximal effect observed was approximately 25% of that observed with ATP or ADP. In the presence of maximally effective concentrations of UTP, the concentration effect curves to 2C1ATP and ADP followed law of mass action interaction at a single site, and their maximal elevation of inositol phosphate accumulation was equivalent to that observed with 2MeSATP and 2MeSADP. The order of potency of adenine nucleotide agonists in the presence of a maximally effective concentration of UTP was consistent with that for interaction with a P2Y1 receptor. Thus, HEK293 cells apparently express two subtypes of P2Y receptors that respond to ADP or ATP in an additive manner: a P2Y1 receptor, which is selectively activated by 2MeSADP, and a P2Y2 receptor, which is selectively activated by UTP.