Urate is a major metabolite of purine metabolism. Urate homeostasis is a complex balance between urate production and renal clearance with marked species differences in the renal handling of urate. To understand urate handling in man we have investigated the transport of urate across polarised monolayers of human proximal tubule cells. Proximal tubule cells were isolated from normal human kidney and grown as monolayers on permeable filter supports for up to 12 days. Under these conditions, PTC monolayers remained differentiated and expressed a full complement of transport proteins including OCT2, OAT1, OAT3, MDR1, MRP4 and BCRP at the appropriate membrane. To characterise urate handling by human proximal tubule cell monolayers, steady state, unidirectional fluxes of [¹⁴C]-urate (45µM) and [³H]-mannitol (5µM) were measured in pair-matched monolayers. In addition at the end of the flux period cell monolayers were excised and the intracellular accumulation of isotope was measured. Data are expressed as the mean ± sem of at least 4 monolayers per condition from a single kidney representative of 3 independent experiments. Statistical comparison was made using a one-way analysis of variance (ANOVA) test and a Dunnett post test. We found a net secretion of urate across proximal tubule cell monolayers. The flux in the secretory direction (Jba) was significantly larger than the corresponding absorptive flux (Jab) of urate (7.7±0.3 v 2.7± 0.9 nmol/cm²/h, n=4, P<0.01) generating a significant net secretion of urate across the monolayer (4.7±0.9 nmol/cm²/h, n=4, P<0.001). Net secretion of urate was abolished in the presence of probenecid or PAH (both at 100µM) at the basolateral membrane. The OAT3-specific substrate estrone-3-sulfate inhibited net secretion by 42±3.2% (n=4, P<0.01) at the basolateral membrane. Net secretion was inhibited 87±6.1% (n=4, P<0.01) by the MRP2/MRP4 inhibitor MK571 (50µM) and by 21.2±7.8% in the presence of the BCRP inhibitor Fumetrimorgin-C (50µM) both at the apical membrane. Consistent with these observations, intracellular accumulation of urate within the monolayer was significantly less in the presence of either probenecid (19.3±3.9µM, P<0.01), PAH 11.4±1.8µM (P<0.01) or estrone-3-sulfate (28.3.±4.4µM, P<0.05) compared to control conditions (45.7±2.5µM). In contrast, inhibition of the apical exit step for urate by MK571 led to significant rise in intracellular urate concentration (82.3±5.6µM v 45.7±2.5µM, P<0.01). Taken together, these data provide evidence for both cellular absorption and secretion of urate across proximal tubule cell monolayers. Urate flux in the secretory direction was significantly larger than in the absorptive direction. Urate secretion was mediated by both OAT1 and OAT3 at the basolateral membrane and predominately by MRP2/MRP4 at the apical membrane.