Extracellularly applied ATP, UTP and UDP induce a transient increase in the intracellular Ca2+ concentration of mammary cells via a P-2U receptor. The P-2U receptor in the mammary tumor cell line MMT060562 was cloned and expressed in the human leukemia cell line K-562. The deduced amino acid sequence of the mammary tumor cell P-2U receptor was 98% homologous with that of mouse NG108-15 cells. It was a member of the superfamily of GTP-binding-protein-coupled receptors. ATP and UTP induced the increase in the intracellular concentrations of Ca2+ and inositol-1,4,5-trisphosphate in both mammary tumor cells and P-2U-receptor-expressed K562 cells. Dose-response curves on the production of inositol-1,4,5-trisphosphate and Ca2+ by ATP and UTP were consistently similar. Injection of GTP enhanced the ATP-induced outward current and injection of GTP gamma S induced a repetitive outward current. Both pertussis and cholera toxins did not affect ATP-induced calcium increase. It was suggested that the P-2U receptor coupled with pertussis- and cholera-toxin-insensitive GTP-binding proteins and activated phosphoinositide turnover.