ABSTRACTBiofuel alcohols have severe consequences on the microbial hosts used in their biosynthesis, which limits the productivity of the bioconversion. The cell envelope is one of the most strongly affected structures, in particular, as the external concentration of biofuels rises during biosynthesis. Damage to the cell envelope can have severe consequences, such as impairment of transport into and out of the cell; however the nature of butanol-induced envelope damage has not been well characterized. In the present study, the effects ofn-butanol on the cell envelope ofEscherichia coliwere investigated. Using enzyme and fluorescence-based assays, we observed that 1% v/v n-butanol resulted in release of lipopolysaccharides from the outer membrane ofE. coliand caused ‘leakiness’ in both outer and inner membranes. Higher concentrations ofn-butanol, within the range of 2% – 10% (v/v), resulted in inner membrane protrusion through the peptidoglycan observed by characteristic blebs. The findings suggest that strategies for rational engineering of butanol-tolerant bacterial strains should take into account all components of the cell envelope.