The characteristics of gentamicin transport have been studied by using cultured kidney epithelial cell line LLC-PK1. The uptake of gentamicin by the LLC-PK1 cells appeared to be linear for 30 min and reached the equilibrium at day 1. Marked stimulation of gentamicin uptake was observed on the development of a confluent cell density, accompanied by the increases of marker enzyme activities and Na+-dependent D-glucose transport in the apical membranes. Gentamicin uptake was inhibited by metabolic inhibitors such as rotenone and 2,4-dinitrophenol, and was inhibited competitively in the presence of other aminoglycosides. Depending on the external calcium concentration, calcium ionophore A23187 stimulated gentamicin uptake, whereas ethylene glycol bis(beta-aminoethyl ether)N,N1-tetraacetic acid, a calcium chelator, inhibited gentamicin uptake. These results suggest that gentamicin uptake by the LLC-PK1 cells may be mediated via specialized transport system, and calcium ion movement may play an important role as a regulatory factor for this transport system.