The effects of gentamicin, an aminoglycoside antibiotic, and verapamil, a calcium channel blocker, on the transport systems in canine renal brush border membrane vesicles were examined. They were tested for their effects on the transport of either a prototypic organic cation, N1-[3H]methylnicotinamide or a prototypic organic anion p-[3H]aminohippurate. They were classified as a substrate for either transport system if the following two criteria were fulfilled: 1) cis inhibition and 2) trans stimulation with concentrative uptake of the prototypic ion. Both gentamicin and verapamil cis inhibited the uptake of the prototypic organic cation N1[3H]methylnicotinamide, with Ki values of 450 and 5 microM, respectively. Similarly, both drugs trans-stimulated N1-methylnicotinamide influx and produced concentrative uptake over the equilibrium value. The results demonstrate that gentamicin and verapamil are substrates for the renal organic cation transport system. We postulate that high-affinity substrates should protect against gentamicin-induced nephrotoxicity.