The impact of the microtubule-disrupting drug nocodazole on renal tubular secretion of organic anions was examined in vitro using proximal tubular masses from teleost fish. Nocodazole reversibly inhibited 20-30% of the tubular accumulation of two model organic anions, p-aminohippurate and fluorescein (FL), by winter flounder tubular masses. However, the drug had no effect on the initial rate of organic anion uptake. Thus it did not reduce transport into the cells at the basolateral membrane, either directly by affecting basolateral organic anion transport proteins or indirectly by altering metabolism or ion gradients. Instead, epifluorescence video microscopy and digital image analysis of killifish tubules showed that nocodazole greatly reduced luminal accumulation of FL and had a smaller effect on cellular dye accumulation. Luminal FL accumulation returned to control levels when tubules were incubated in drug-free medium. Confocal fluorescence microscopy confirmed the marked reduction in luminal FL concentration and demonstrated that intracellular punctate FL accumulation was also markedly reduced. Finally, immunohistochemistry with an anti-tubulin antibody showed that the concentrations of nocodazole used in the above experiments reversibly disrupted microtubules within renal epithelial cells. These data indicate that a component of organic anion secretion in teleost proximal tubule is dependent on an intact microtubular network.