A major portion of tetracycline accumulation by susceptible bacterial cells is energy dependent. Inner membrane vesicles prepared from susceptible Escherichia coli cells concentrated tetracycline 2.5 to 5 times above the external concentration when the electron transport substrate D-lactate or reduced phenazine methosulfate was added. This stimulation was reversed by cyanide, 2,4-dinitrophenol, and carbonyl cyanide m-chlorophenyl hydrazone. These vesicles data showed that proton motive force alone could energize tetracycline uptake. The lactate-dependent uptake had a pH optimum of 6.9 and a magnesium optimum of 1 mM and was not saturable up to 400 microM tetracycline. Although the vesicles were not as active as cells in concentrating tetracycline, they were less active to a similar extent in concentrating tetracycline, they were less active to a similar extent in concentrating proline, the transport of which is known to be solely proton motive force dependent. Therefore, we concluded that the active uptake of tetracycline in susceptible cells was largely, if not solely, energized by proton motive force.