Polyclonal stimulation of normal mouse spleen cells by lipopolysaccharide (LPS) from Salmonella typhimurium resulted in the generation of a factor which was capable of suppressing the humoral immune response in vitro. LPS effectively induced the release of the inhibitory material into the supernatants of these cultures within 24 hr. The suppressive mediator, which was similar in properties to the antigen-generated, transiently-acting soluble suppressor (TASS) reported earlier, partially abrogated (by 30–80%) the anti-sheep erythrocyte plaque-forming cell response when added to test cultures ~20 hr prior to assay for direct hemolytic plaques. Although LPS, in submitogenic doses, also was effective in depressing the in vitro hemolysin response, the inhibitory activity of residual mitogen present in the test supernatants, and that of the LPS-induced factor, were shown to be different. By use of antisera and complement treatment to selectively deplete spleen cell populations of T or B lymphocytes, it was demonstrated that B cells were essential for production of the suppressive mediator.