Growth of a beta-oxidation-negative (fadE) strain of Escherichia coli in liquid culture with exogenous palmitate leads to the accumulation of 16:0/16:0 molecular species of phospholipid resulting in a temporary decrease in growth rate and lysis of a variable fraction of the cell population. Under the same growth conditions, this behavior is not seen in the closely related fadE+ parent strain which accumulates more modest levels of 16:0/16:0 together with 16:0/14:0. Based on differential scanning calorimetric measurements, 75.8 and 17.5% of the lipids from 16:0-enriched fadE and fadE+ cells, respectively, were found to be in the gel state at the growth temperature. Kinetic studies reveal the translocation of 16:0/16:0 molecular species from inner to outer membrane delays briefly the accumulation of this species in the inner membrane. This extreme and deleterious change in membrane-lipid composition precludes cloning of the fadE strain on solid media containing 16:0 and, therefore, these conditions provide the basis for selection of mutants altered in the mechanisms which determine the synthesis or accumulation of membrane lipid. Three such mutants are described which display alterations in the normal distribution of molecular species.