AbstractFor intact cells of A. calcoaceticus 69V susceptibility to hydrophobic agents (antibiotics, dyes) was established. The composition of its outer membrane and comparison with that of a reference strain, A. calcoaceticus CCM 5593 with a blocked hydrophobic pathway, gave no indication of phospholipid bilayer domains as the structural basis of these permeability characteristics. The outer membrane composition together with the data of time‐resolved fluorescence anisotropy measurements is indicative of a high state of order of the hydrocarbon region.A. calcoaceticus 69V releases lipopolysaccharide (LPS)‐rich membrane vesicles into the growth medium when grown on a hydrophobic carbon source. While the cells contain both R‐form and S‐form LPS, the LPS released with the vesicles is exclusively of the R‐type. The same selectivity with respect to LPS composition was observed when LPS was removed from intact cells by EDTA‐NaCl treatment which leads to a break‐down of the barrier to hydrophobic agents in A. calcoaceticus CCM 5593.We propose that due to its physical properties, R‐form LPS forms tightly packed structures within the membrane which, under certain conditions, become destabilized and liberated into the surrounding medium. As a consequence, a disturbance of the highly ordered lateral molecular arrangement might lead to altered permeability properties of the outer membrane as suggested in one of the two alternative models existing to explain permeability changes observed in deep rough mutant strains of Enterobacteriaceae.