Gentamicin uptake and toxicity was studied in a nontransformed cell line obtained from the S1 segment of the proximal tubule epithelium of a transgenic mouse. Cytotoxicity was assayed using the dye 3-(4,-5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Gentamicin uptake was assayed by a fluorescence polarization assay. No differences in toxicity were found among cells incubated for 4 hours in complete culture medium, enriched Kreb's buffer alone, or enriched Krebs' buffer with added 300 micrograms/mL gentamicin, 0.5 mmol/L L-lysine, or gentamicin plus L-lysine. Uptake of 300 micrograms/mL gentamicin was minimal at zero time and increased as a function of time. Uptake of gentamicin at 4 hours was positively correlated with medium gentamicin concentration. Addition of 0.5 mmol/L L-lysine inhibited uptake of 300 micrograms/mL gentamicin 38.9 +/- 10.2%. No other amino acid, including D-lysine or arginine, significantly changed gentamicin uptake. The authors conclude that gentamicin and L-lysine share a specific uptake mechanism located in the apical membrane of renal proximal tubule cells.