The uptake of chloramphenicol by susceptible strains of Escherichia coli and Pseudomonas aeruginosa was measured as the depletion of 14C-chloramphenicol from the supernatant of centrifuged cultures. Chloramphenicol did not bind to non-growing cells or isolated cell envelopes. Chloramphenicol was recovered from cells in an unchanged form and was intracellularly concentrated several times above external concentrations. The net accumulation of the drug was reduced by an inhibitor of electron transport, by an oxidative phosphorylation uncoupler, by an inhibitor of high energy phosphate synthesis, and by lowering the temperature to + 15°C. The initial uptake of drug was saturated at 1.98 mM chloramphenicol in the medium. A 100-fold excess of each of the unlabelled isomers: L-threo, D-threo, and L-erythro chloramphenicol in cultures of either strain effectively reduced the uptake of 14C-chloramphenicol. These results indicate that chloramphenicol enters Gram-negative bacteria by means of an energy-dependent proces
