The cells involved in the stimulatory effect of bacterial lipopolysaccharide (LPS) on the induction of immunoglobulin (Ig) production by rabbit spleen cells cultured in the absence of antigen has been analyzed. Addition of LPS caused a several-fold enhancement of both DNA synthesis and Ig production. These enhanced activities were not significantly affected by depletion of adherent cells in the spleen cell population. Although inactivation of splenic T cells by anti-thymocyte serum (ATS) treatment did not affect the enhancement of DNA synthesis due to LPS, such treatment did adversely affect the enhancement of Ig production by LPS. Furthermore, the enhancement of Ig production of ATS-treated spleen cells by LPS was found to be dependent on the number of thymocytes added. In addition, the prior incubation of ATS-treated spleen cells with LPS resulted in effective enhancement of Ig production when such ATS-treated spleen cells and thymocytes were combined after removal of LPS. An identical experiment, except that thymocytes instead of ATS-treated spleen cells received the prior incubation with LPS, did not result in enhancement of Ig production. Finally, the enhanced Ig production due to LPS was inhibited by hydroxyurea, a known inhibitor of cellular DNA synthesis. The relationship between the mitogenic activity of LPS on B cells, the regulatory function of T cells, and the enhancement of Ig production by LPS is discussed in relation to the contrasting reports concerning the cellular target of LPS.