The mechanisms driving organic anion transport from cell to lumen were studied in intact killifish proximal tubules using fluorescence microscopy. Three fluorescent substrates were used as follows: 1) fluorescein (FL); 2) carboxyfluorescein (CF), generated intracellularly from carboxyfluorescein diacetate (CFDA); and 3) bimane-S conjugates, generated intracellularly by conjugation of monochlorobimane (MCB) with glutathione (GSH) and subsequent metabolism. The latter two substrates bypassed the basolateral uptake mechanism, allowing direct study of luminal transport mechanisms. At steady state, for all three substrates, luminal fluorescence was two to three times higher than cellular fluorescence. With FL as substrate, addition of p-aminohippurate (PAH) or probenecid to the incubation medium reduced cellular and luminal fluorescence to roughly the same extent. With CFDA or MCB as substrate, PAH and probenecid only slightly reduced cellular fluorescence but greatly reduced luminal fluorescence. MCB blocked transport of FL from cell to lumen; CFDA blocked transport of bimane-S conjugates from cell to lumen. Finally, depolarizing tubule cells with high-potassium medium did not affect the steady-state lumen-to-cell distribution of FL, CF, or bimane-S conjugates. These results show that organic anion transport from cell to lumen is mediated and uphill but not sensitive to the electrical potential difference across the luminal membrane.