An analysis of which component of lipopolysaccharide, the lipid or the polysaccharide, is mitogenic for mouse B-lymphocytes has been performed. A purified glycolipid derived from a rough mutant of Salmonella minnesota (R595) that does not contain any o-polysaccharide at all is more mitogenic than an intact lipopolysaccharide derived from a smooth strain of S. minnesota. Results using fractions produced by several different chemical modifications of whole lipopolysaccharide confirm this result. Acid hydrolysis separates lipopolysaccharide into two components. The lipid fraction is mitogenic, whereas the polysaccharide fraction is not. Those procedures which degrade or modify only the lipid moiety while preserving the antigenic integrity of the polysaccharide also destroy mitogenicity. These include alkaline hydrolysis and deacylation by a more specific treatment with potassium methylate. The lipid preparations are fully active on highly purified B-lymphocyte populations (prepared by anti-theta antiserum and complement), whereas they have no effect on highly purified T-lymphocyte populations (prepared by anti-immunoglobulin and complement). These data demonstrate that the lipid moiety of endotoxin is the B-lymphocyte mitogen, whereas the polysaccharide has no demonstrable mitogenic activity.