Renal handling of NS-49, which is an organic cation and a chiral compound, was investigated in rats, rabbits and dogs. Renal clearance (Cl re ) of NS-49 was 3.4-fold the glomerular filtration rate (GFR) in the rat in vivo study. The clearance ratio (Cl re /GFR) approached unity during cimetidine infusion. Change in the urine flow rate or urinary pH did not affect the Cl re of NS-49. The stop-flow patterns of NS-49 in the rabbits and dogs showed a secretion peak in the proximal tubules. On concomitant administration of cimetidine, the secretion peak disappeared, the stop-flow pattern showing neither a secretion nor reabsorption peak. These findings indicate that in these species NS-49 undergoes glomerular filtration and extensive proximal tubular secretion, but little reabsorption. A transport mechanism study of NS-49 in brush-border membrane vesicles (BBMVs) isolated from rat kidney cortex showed that it is transported via the carrier-mediated H + /organic cation antiport system. In the rat renal clearance studies (in vivo) tubular secretion of NS-49 was significantly inhibited by quinine (p B 0.01) but not by quinidine. Transport studies done with rat BBMVs (in vitro) also showed quinine to be more potent than quinidine in inhibiting NS-49 uptake. These results indicate that stereoselective interaction occurs in active renal tubular secretion.