A key function of biological membranes is to exclude toxic small molecules while allowing influx of nutrients. Cells achieve this by controlling the composition of different types of proteins and lipids within the membrane by a process called membrane biogenesis. We have recently proposed a strategy to identify genes involved in membrane biogenesis in Gram-negative bacteria such as Escherichia coli by selecting for suppressors of mutations that render the outer membrane (OM) leaky. We predicted that different small molecules could select different suppressors because mutations that answer a specific selection will correct the membrane permeability defect to different degrees depending on the structure of the small molecule. We have tested this hypothesis by selecting for resistance to bile acids in an imp4213 strain, which contains a compromised OM owing to a defect in lipopolysaccharide biogenesis. We report here that a suppressor mutation in yaeT , which specifies an essential protein involved in the assembly of beta-barrel proteins in the OM, confers resistance to a specific subset of bile acids in the imp4213 strain. YaeT is conserved from bacteria to man because it is involved in OM biogenesis in mitochondria and chloroplasts. These results demonstrate that structurally different toxic small molecules select different, and highly specific, genetic solutions for correcting membrane-permeability defects. The remarkable chemical specificity of the imp4213 suppressors provides insights into the molecular nature of the OM permeability barrier.