Transgenic maize expressing the non-toxic B subunit of the Escherichia coli heat-labile toxin (LT-B) in seed has proven to be an effective oral immunogen in mice. Currently, there is considerable concern over accidental consumption of transgenic maize expressing LT-B by humans and domestic animals. While consuming maize-expressed LT-B appears to have no toxic side effects, we have yet to define nonimmunogenic levels of transgenic LT-B when ingested. We also intend to determine if accidental exposure to LT-B could affect a later response to a vaccine containing LT-B as either an antigen or a carrier.;Our first goal was to determine the largest dose of LT-B orally administered in mice that does not result in a measurable immune response. Mice were fed low doses of LT-B intermittently (days 0, 7 and 21) resembling vaccine dose scheduling. To determine the effects of previous exposure on vaccine administration, we fed mice intermittently or daily for 28 days to resemble two distinct inadvertent exposure scenarios. We subsequently boosted all mice with vaccine-level doses of LT-B. To determine immune responses, serum and fecal pellets were collected weekly for measurement of LT-B-specific antibodies.;Mice fed 0.02 microg LT-B intermittently demonstrated immune priming in 62.5% of the animals. Mice that were fed ≤0.002 microg LT-B showed no increase in specific antibody nor did they demonstrate immune priming, thus indicating that 0.002 microg LT-B was the highest nonimmunogenic dose tested. Mice that were exposed to maize-derived LT-B, whether daily or intermittently, generate dose-dependent antibody responses to LT-B. All animals that had been previously exposed to LT-B by either intermittent or daily feeding responded strongly to the vaccine-like booster doses, indicating that mice orally exposed to LT-B do not develop oral tolerance to LT-B. Thus, inadvertent oral exposure to LT-B may not negatively impact future vaccinations containing LT-B as either an antigen or carrier.