Accumulation of gentamicin by gentamicin-susceptible Staphylococcus aureus was examined by using the ionophorous antibiotic, valinomycin and the proton-translocating ATPase inhibitor N,N′-dicyclohexylcarbodiimide. The effects of these inhibitors on the transmembrane electrical potential (Δψ) were determined by measuring the equilibrium distribution of the tetraphenylphosphonium ion. The results indicate a direct correlation between Δψ and the extent of gentamicin uptake. However, under conditions where a significant Δψ existed across the plasma membrane, uptake of gentamicin was negligible. A threshold Δψ may thus be required to initiate gentamicin uptake. The proposed threshold Δψ appears to vary depending upon the external concentration of gentamicin.