Six mutants of Salmonella typhimurium LT2 with defects in the heptose region of the lipopolysaccharide (LPS) ("rough" mutants) were more sensitive to the growth-inhibitory effects of erythromycin, bacitracin, vancomycin, novobiocin, kanamycin, and cloxacillin and of deoxycholate than smooth strains, but less sensitive to tetracycline and ampicillin. In general, growth of the three rough mutants of chemotype Rd2, which lack the distal but not the proximal heptose unit in the LPS, was less inhibited than the three mutants of chemotype Re, which are heptose-deficient. In addition, inhibition of uracil-1-14C incorporation in the presence of actinomycin D and spheroplast formation in the presence of lysozyme occurred in the rough mutants without ethylenediaminetetraacetate (EDTA) treatment of the cells, while actinomycin D and lysozyme were effective on smooth strains only after EDTA treatment. Since the major part of the LPS is in the outer membrane of the cell envelope, and since the target of the toxic agents used is located inside this layer, these data indicate that the carbohydrate part of the LPS component of the outer membrane is an essential part of a barrier layer preventing penetration of large molecules.