Glycoprotein 330 (gp330) is an endocytic receptor expressed in the renal proximal tubules and some other absorptive epithelia, e.g., in the inner ear. The present study shows that the antifibrinolytic polypeptide, aprotinin, and the nephro- and ototoxic antibiotics, aminoglycosides, and polymyxin B compete for binding of 125I-urokinase-plasminogen activator inhibitor type-1 complexes to purified rabbit gp330. Half maximal inhibition was measured at 4 microM for aprotinin, 50 microM for gentamicin, and 0.5 microM for polymyxin B. Drug binding to gp330 was validated by equilibrium dialysis of [3H] gentamicin-gp330 incubations and binding/uptake studies in rat proximal tubules and gp330-expressing L2 carcinoma cells. Analyses of mutant aprotinins expressed in Saccharomyces cerevisiae revealed that basic residues are essential for the binding to gp330 and renal uptake. The polybasic drugs also antagonized ligand binding to the human alpha 2-macroglobulin receptor. However, the rapid glomerular filtration of the drugs suggests kidney gp330 to be the quantitatively most important target. In conclusion, a novel role of gp330 as a drug receptor is demonstrated. The new insight into the mechanism of epithelial uptake of polybasic drugs might provide a basis for future design of drugs with reduced toxicity.