Several responses to bacterial endotoxin (lipopolysaccharide, LPS) LPS) have been examined in germfree (GF), Escherichia coli monoassociated (Mono), and conventional (Conv) mice, including immunogenicity, mitogenicity, and lethality. GF mice exhibited significant antibody responses to LPS at low concentrations and over a broad dose range. However, the development of this response was considerably different from Conv mice, i.e., Conv mice exhibited peak responses at 14 days after LPS administration, and titers decreased over the next 2 weeks. GF mice manifested moderate responses by 2 weeks and attained highest titers at 4 weeks, which were greater than those obtained with Conv mice. To assess the influence of a Gram-negative bacterial strain on the host's responsiveness of LPS, GF mice were monoassociated with E. coli K235 and compared to GF and Conv mice with regard to immunogenic, mitogenic, and toxic effects of endotoxin. When TNP-LPS immunogen was administered by either the i.p. or gastric route, the highest numbers of splenic anti-TNP PFC occurred in GF mice, whereas the Mono animals exhibited intermediate responsiveness. This pattern was also dose dependent since GF mice responded at TNP-LPS concentrations that were minimally immunogenic for Mono mice and nonimmunogenic for Conv mice. These differences were also reflected in anti-TNP responses in vitro. The splenic mitogenic responses to PHA, Con A, PWM, and LPS in these three groups were assessed, and no differences were noted with the various lectins. However, both GF and Mono mice yielded higher responses to LPS than was observed in Conv mice. Monoassociated mice were also intermediate in their sensitivity to LPS-induced lethality; GF mice were approximately 5-fold more resistant than Conv mice. The results of this study demonstrate that GF mice in contrast to Conv mice exhibit greater in vitro mitogenic and immunogenic responses and are more refractory to endotoxin.