Our previous study has demonstrated the activation of calcium-dependent Cl(-) secretion through P2Y(2) receptors by extracellular nucleotides. To evaluate the contribution and involvement of other receptor subtypes to this physiological response, the purpose of this study was to investigate the regulation of intracellular calcium concentration ([Ca(2+)](i)) by different purinergic agonists in cultured rat Sertoli cells. Cultured epithelia of Sertoli cells from immature rats were grown on glass coverslips and the purity was confirmed by immunohistochemistry. Intracellular calcium concentration was monitored by microspectrofluorimetric technique using calcium sensitive fluorescent dye Fura-2. Results showed that UTP and ATP consistently increased [Ca(2+)](i). Adenosine, AMP and 2-methylthio-ADP (2-MeSADP) failed to elicit a discernible response. Cross desensitization experiments suggested that the responses to UTP and ATP were mediated by a common receptor population. This is consistent with the presence of receptors belonging to the P2Y(2) subclass. Sertoli cells also responded to 2-methylthio-ATP (2-MeSATP) and ADP but not 2-methylthio-ADP (2-MeSADP) in a concentration-dependent manner. Moreover, the ATP-or UTP-pretreated epithelia continued to respond to ADP, indicating that these cells express at least one additional receptor population that allowed nucleotides to increase [Ca(2+)](i). Apart from the P2Y receptor agonists, the P2X(4) and P2X(7) agonist, 2' and 3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), also evoked [Ca(2+)](i) increases in rat Sertoli cells. Messenger RNA transcript corresponding to P2Y(1), P2Y(2), P2X(4) and P2X(7) receptors genes were detected using RT-PCR. Taken together, these data indicate that both metabotropic receptors and ionotropic receptors are present in rat Sertoli cells through which extracellular nucleotides can act.