The translocation of lipids from the inner to the outer membrane of Escherichia coli has been investigated by pulse-chase experiments. After a pulse with [2-3H]glycerol, the specific activity of the newly synthesized [3H]phosphatidylethanolamine was 5 times greater in the inner than in the outer membrane. During the chase, [3H]phosphatidylethanolamine was translocated to the outer membrane. At 37 degrees C, the half-life for translocation was 2.8 min. This rate was not influenced by alteration in the cellular growth rate at 37 degrees C. Altering the cellular growth temperature had a pronounced effect on the rate of phosphatidylethanolamine translocation. Energy inhibitors that deplete the protonmotive force markedly inhibited the translocation. Translocation was not affected by inhibitors of ATP, protein, or lipid synthesis. Phosphatidylglycerol and cardiolipin are transfocated very rapidly, with half-lives shorter than 30 sec.