P-Aminohippuric acid (PAH) transport was investigated in brush border vesicles isolated from renal proximal tubules of the winter flounder. Three characteristics of carrier-mediated transport were demonstrated: unlabeled PAH inhibited the uptake of [3H]PAH; [3H]PAH efflux from the vesicles was stimulated in the presence of unlabeled PAH in the extravesicular medium; and PAH influx was inhibited by 2,4-dinitrophenol (DNP) and 4-acetamido-4''-isothiocyano-2,2''-disulfonic stilbene (SITS). D-Glucose plus a Na gradient stimulated PAH uptake, as did a K2SO4 gradient plus valinomycin, suggesting that PAH is transported as an anion. PAH uptake into a membrane fraction containing mainly basal-lateral plasma membranes exhibited a larger inhibition by probenecid but a smaller inhibition by unlabeled PAH and SITS. Carrier-mediated transfer of PAH driven by the electrochemical potential difference for PAH is demonstrated in the brush border membrane of the flounder kidney.