Previous work has shown that the indigenous gut microflora in mice plays a protective role against Escherichia coli heat-labile enterotoxin (LT)-mediated abrogation of oral tolerance to an unrelated co-ingested protein. To assess potential protection by human gut microflora, we studied the effect of human gut microflora in a murine model. Oral tolerance was studied in adult gnotobiotic mice (i.e. ex-germ-free mice) colonized with the entire human fecal microflora and orally administered once with LT and ovalbumin. Systemic suppression of IgG, IgG1, IgG2a, and IgE antibody responses was assessed by ELISA. Both specific IgG subclasses and IgE hyporesponsiveness was induced in LT + ovalbumin-fed gnotobiotic mice, indicating that the human gut microflora can protect against the LT-mediated abrogation of oral tolerance. However, as confirmed with mouse gut microflora, this protective effect only occurs when the gut microflora is associated from birth on. Colonization of germ-free mice with a single bacterial strain, E. coli, predominant in the human and mouse gut microflora in the neonatal period, showed that this strain alone did not induce protection. These results supported the hypothesis that the natural establishment of the gut microflora in neonates crucially influenced resistance to LT-mediated abrogation of oral tolerance by reinforcing suppression of both T helper type 1- and T helper type 2-controlled responses, and suggested that sequential bacterial colonization of the gut rather than a single bacterial species may be involved in this phenomenon.