Sodium vanadate is a potent inhibitor of Na-K-adenosine triphosphatase. p-Aminohippurate (PAH) and tetraethylammonium accumulation in rat renal cortical slices was inhibited by vanadate in a dose-dependent manner at medium vanadate concentrations from 10(-6) to 10(-3) M. Inhibition was reversible at vanadate concentrations less than 1.5 x 10(-5) M. The slice content of vanadium (7.5-325 micrometer V/g wt. of tissue) was linearly related to medium vanadate concentrations ranging from 10(-5) to 10(-3) M. The ability of slices to generate glucose and ammonia was not impaired by medium vanadate concentrations up to 5 x 10(-4) M, a concentration that maximally inhibited organic ion accumulation. Increasing medium K+ concentrations potentiated vanadate inhibition of PAH accumulation which correlated with inhibition of sodium pump activity, as determined by 42K+ uptake. Intraperitoneal administration of vanadate (1 or 5 mg V/kg) to rats produced a profound diuresis and natriuresis during the 1st hr. Inhibition of PAH accumulation of renal slices from these rats was related to tissue vanadium concentrations. These data suggest that vanadate exerts its action on proximal tubule transport of PAH via inhibition of Na-K-adenosine triphosphatase.