In astrocytes, raising intracellular Ca2+ concentration is a principal mechanism for transducing extracellular signals following activation of cell-surface receptors. Receptors that may be activated by purine nucleotides, P2 receptors, are known to be expressed by astrocytes from dorsal spinal cord; these astrocytes express two distinct subtypes of P2 receptor, P2Y and P2U. A main goal of the present study was to determine the intracellular signalling pathways mediating the Ca2+ responses produced by stimulating these receptors. Experiments were done using cultured astrocytes from rat dorsal spinal cord. Ca2+ responses were evoked by 2-methylthio-ATP or UTP, nucleotides previously shown to selectively activate P2Y and P2U receptors, respectively, in these cells. P2Y- and P2U-evoked Ca2+ responses were found not to depend upon extracellular Ca2+ and were blocked by thapsigargin, a Ca2+-ATPase inhibitor known to deplete inositol 1,4,5-triphosphate-sensitive Ca2+ stores. Intracellular application of the inositol 1,4,5-triphosphate-sensitive receptor antagonist, heparin, or of the G-protein inhibitor guanosine 5'-O-(2-thiodiphosphate), blocked the P2Y- and P2U-evoked Ca2+ responses. Moreover, the responses were prevented by the phospholipase C inhibitor, U-73122, but were unaffected by the inactive analogue, U-73343. These results indicate that P2Y and P2U receptors on dorsal spinal astrocytes are linked via G-protein coupling to release of intracellular Ca2+ via the phospholipase C/inositol 1,4,5-triphosphate pathway. When we assessed the releasable pools of intracellular Ca2+, by repeated agonist applications in zero extracellular Ca2+, we found that the pool accessed by activating P2U receptors was only a subpool of that accessed by activating P2Y receptors. This implies that there are separable inositol 1,4,5-triphosphate-releasable pools of Ca2+ in dorsal spinal astrocytes and that these may be differentially released by activating distinct metabotropic P2 receptors. This differential release of Ca2+ may be important for physiological as well as pathophysiological events occurring within the spinal cord.