Dimethylsuberimidate cross-linking has been used to determine the arrangement of phosphatidylethanolamine (PE) molecular species in Escherichia coli membranes. No large deviations from random mixing were found in wild-type strain AB1623, either in whole cells or in extracted lipids which were reconstituted into multilamellar vesicles. These results suggest, first, that there is little difference in the PE molecular species composition of the three lipid monolayers (the inner and outer monolayers of the inner membrane and the inner monolayer of the outer membrane) which contain significant amounts of PE. Secondly, the results suggest that the molecular species within each monolayer and in the extracted lipids are arranged close to randomly with no tendency for like molecular species to cluster. E. coli strain L8-2, which has a defect in beta-oxidation and a temperature-sensitive mutation in total fatty acid synthesis, was grown on cis-vaccenate (cis-11,12- octadecenate) to enrich the cells in divaccenoyl PE. Again, in whole cells or in lipids extracted from whole cells and reconstituted into multilamellar vesicles, the species were close to randomly arranged. However, a consistent, slight tendency of divaccenoyl species to pair with like species as compared to pairing with the second most common species, vaccenoyl, palmitoleoyl PE, was noted in both extracted lipids and in whole cells.