Our past studies have shown that bacterial lipopolysaccharide (LPS) affects gut-associated lymphoreticular tissue (GALT) for both induction of IgA responses to orally administered thymic-dependent (TD) antigens and systemic unresponsiveness (oral tolerance). In the present study, we have directly examined the effects of LPS on oral tolerance induction by use of germfree (GF) mice, which have not previously experienced LPS. GF BALB/c mice do not respond as well to systemically administered sheep erythrocytes (SRBC) as their conventional (Conv) counterparts; however, both primary and secondary responses to SRBC were seen in GF mice. Furthermore, anti-SRBC plaque-forming cell (PFC) responses were induced in GF spleen cell cultures, and these responses could be enhanced with the adjuvants LPS and muramyldipeptide (MDP); however, these in vitro responses were significantly lower (p ≤ 0.01) than those observed in cultures from Conv mice. Daily oral administration of SRBC by gastric intubation (GI) to GF mice for 2 wk or longer, primed these animals for anamnestic responses when systemically (i.p.) challenged with SRBC. The same treatment of Conv mice induced oral tolerance. Lack of oral tolerance induction in GF mice was not strain related, because GF BALB/c and C3H/HeN inbred and Swiss outbred mice GI with SRBC for 2 wk and subsequently immunized i.p. with SRBC gave significant IgG1, IgG2, and IgA anti-SRBC PFC responses, whereas identically treated Conv mice from these three strains were unresponsive. Prior oral treatment of GF BALB/c mice with different doses of LPS followed 5 days later by GI with SRBC resulted in oral tolerance induction. Ten micrograms or more of LPS were sufficient for conversion of mice to full sensitivity to oral tolerance induction, and 1 μg of LPS diminished the ability of these mice to respond to antigen. Oral tolerance in LPS-treated GF or Conv BALB/c could be reversed by administration of LPS with the i.p. challenge of SRBC; LPS also reversed unresponsiveness of spleen cell cultures from these mice to SRBC in vitro. The cellular basis for oral tolerance in LPS-treated GF and Conv mice may be due to regulatory T suppressor (Ts) cells, because prior treatment of spleen cells from these mice (given SRBC by GI for 2 wk) with anti-Lyt-2 and complement abrogated unresponsiveness to SRBC. These results suggest that LPS from the gut is an important determinant for immune responses or oral tolerance to orally encountered TD antigens, and this may be mediated by a direct effect of LPS on GALT T cells. The relationship between LPS influence on GALT T cells, which determined whether the host will respond (or fail to respond) to TD antigen, is discussed.