Wild-type strains of Escherichia coli K-12 adsorb gentian violet to the cell surface, but the dye is not transported into the cytoplasm. However, when some mutants that have an altered outer membrane are exposed to gentian violet, the dye is also found in the ribosomal fraction. The transport into the cytoplasm is inhibited at 0 C and requires that the concentration of gentian violet exceeds a threshold value. The initial rate of uptake as well as the amount of gentian violet found in the cytoplasm increases with the concentration of the dye in the medium. The rate of transport of the dye into the cytoplasm is much lower for stationary mutant cells than for exponentially growing cells. The rate of uptake into the cytoplasm increases with increasing deficiency of carbohydrate in the lipopolysaccharide (carbohydrate content lpsB > lpsA > galU). However, other components are also responsible for the barrier since an envA mutant which is not altered in the lipopolysaccharide carbohydrates show an extremely rapid uptake of the dye. The rate of uptake for the envA mutant was the highest found and the same as that of spheroplasts. Growth in the presence of agents affecting the murein sacculus, e.g., lysozyme and sublethal concentrations of penicillin, increased the rate of uptake of gentian violet. Brief treatments with tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid drastically impaired the barrier function. Inhibition of protein synthesis by chloramphenicol also opened the barrier to gentian violet. In conclusion, the outer part of the bacterial envelope is a penetration barrier for gentian violet and probably also for other substances. The lipopolysaccharide, the murein and also other components are important for the function of this barrier. Resistance to gentian violet was found to be inversely correlated to the rate of penetration of the dye into the cytoplasm.