The in vitro immune responsiveness of cells from germfree (GF) mice was investigated by employing lectins and endotoxin. Splenocytes from GF, when compared with those from conventional (Conv), mice manifested similar responses to the T lymphocyte mitogens, PHA and Con A. When a purified Escherichia coli K235 lipopolysaccharide (LPS) was tested, GF splenocytes gave 2- to 3-fold greater stimulation than those observed with Conv spleen cells. This difference in mitogenic responsiveness to LPS was not dependent upon culture conditions or greater numbers of B lymphocytes in GF spleen. Purified lipid A, and to a lesser extent lipid A-associated protein, elicited higher responses with B cells from GF mice. Lower responses to LPS were observed after conventionalization of GF mice with indigenous BALB/c flora. Similar results were obtained when a second LPS effect was measured, anti-TNP PFC responses after TNP-LPS immunization. To determine the nature of the cell(s) involved in this suppression, macrophages (Mphi) and purified lymphocytes were derived from GF and Conv spleens, and various cell combinations were tested for their mitogenic and immunogenic responsiveness to LPS. No apparent differences in anti-TNP PFC and mitogenic responses could be detected between GF and Conv Mphi when tested with T and B cells derived from GF mice. However, lower responses were always seen with Conv T and B cells and either GF or Conv Mphi, suggesting a lymphocyte origin for suppression. Purified T cells from either Conv spleen or Peyer's patches significantly reduced mitogenic and TNP-LPS immune responses in GF spleen cell cultures. The presence of T suppressor cells was further supported by the observation that GF and nude mice exhibited higher responses to TNP-LPS. This evidence taken together suggests that the normal Gram-negative gut flora induces, possibly via lipid A, the production of a T cell population that regulates B cell responses to bacterial endotoxin.