We constructed a full-length human P2X5 purinoceptor cDNA by incorporating a sequence corresponding to exon 10, which is missing in cDNAs cloned previously from human tissues. We studied the functional properties by patch-clamp recording and fluorescence imaging after expression in human embryonic kidney 293 cells. ATP (1-100 microM; half-maximal current at 4 microM) elicited inward currents at -60 mV; these persisted during brief (2 s) applications but declined during longer applications. The peak current was dependent on the holding potential and showed little rectification; however, both the desensitization during the application and the decline in the current when ATP was washed out were slower at +30 mV than at -60 mV. 2',3'-O-(4-Benzoyl)-benzoyl-ATP and alphabeta-methylene-ATP mimicked the action of ATP (half-maximal concentrations 6 and 161 microM, respectively). The currents were inhibited by suramin, pyridoxal-5-phosphate-6-azo-2',4'-disulfonic acid and Brilliant Blue G, with half-maximal inhibition at 3, 0.2, and 0.5 microM, respectively; 2',3'-O-(2',4',6'-trinitrophenol)-ATP (1 microM) was ineffective. Removing divalent cations did not significantly alter ATP concentration-response curves. Reversal potential measurements showed that the human P2X5 receptor was permeable to calcium (PCa/PNa = 1.5) and N-methyl-d-glucamine (NMDG) (PNMDG/PNa = 0.4); it was also permeable to chloride (PCl/PNa = 0.5) but not gluconate (Pgluc/PNa = 0.01) ions. The permeability to NMDG developed as quickly as the channel opened, in contrast to the P2X7 receptor where the NMDG permeability develops over several seconds. Cells expressing human P2X5 receptors also rapidly accumulated the propidium dye YO-PRO-1 in response to ATP.