Previous studies with p-aminohippurate (PAH) and fluorescein (FL) have shown that cellular uptake and tubular secretion of organic anions is driven by indirect coupling to sodium. Here we used killifish proximal tubules and laser-scanning confocal microscopy to study the transport of a larger organic anion, fluorescein-methotrexate (FL-MTX, mol mass 923 Da). When tubules were incubated in medium containing 2 microM FL-MTX, dye accumulated in both cells and tubular lumens. At steady state, luminal fluorescence was 4-5 times higher than cellular fluorescence. Ouabain (0.1 mM) did not affect cellular or luminal fluorescence, and replacement of medium sodium by N-methylglucamine had only a modest effect; preincubation with glutarate had no effect. KCN did not affect cellular uptake but abolished secretion into the lumen. Uptake and secretion of FL-MTX were inhibited by micromolar concentrations of other organic anions (MTX, folate, probenecid, bromocresol green, bromosulfophthalein), but 1 mM PAH had a relatively small effect. FL-MTX secretion into the lumen was inhibited by leukotriene C4, cyclosporine A, and verapamil, none of which affected FL transport. Thus a substantial component of FL-MTX secretion is Na independent and ouabain insensitive. Both the basolateral and luminal steps in the Na-independent pathway differ from those usually associated with FL and PAH secretion.