For the first time the transmembrane movement of an endogenously synthesized phospholipid across the inner membrane of E. coli is reported. [14C]phosphatidylethanolamine (PE) was biosynthetically introduced into inner membrane vesicles from the PE-deficient strain AD93, by reconstitution with the enzyme phosphatidylserine (PS) synthetase. Upon addition of wild type cell lysate containing PS synthetase, and the metabolic substrates CTP and [14C]serine to inside-out vesicles from AD93, [14C]PS was synthesized, which was for the most part converted into [14C]PE. [14C]PE was introduced in right-side out vesicles by enclosing PS synthetase and CTP in the vesicle lumen and adding [14C]serine. The newly synthesized [14C]PE immediately equilibrated over both membrane leaflets (t1/2 less than one min), as determined by its accessibility toward the amino-reactive chemical fluorescamine. In both inside- out and right-side out vesicles, a 35-65% distribution was found of the newly synthesized PE over the cytoplasmic and periplasmic leaflet, respectively. The transport process of PE was not influenced by the presence of ATP or the proton motive force in inside out vesicles. Pretreatment of both types of vesicles with sulfhydryl reagents, or of right-side out vesicles with proteinase K, did not affect the rate and extent of the transmembrane distribution of the newly synthesized PE.