The respective contributions to protection of the route and dosage of primary and booster immunizations with Escherichia coli heat-labile enterotoxin were evaluated in rats. The degree of protection was determined by challenge with toxin and viable bacteria in ligated ileal loops, and the serum antitoxin response was assayed by enzyme-linked immunosorbent assay. Primary immunization was effective only when given by the parenteral route. The degree of protection was enhanced a fivefold dosage increase in the primary parenteral immunization in rats given constant dosages of booster immunizations either parenterally or perorally, but not by further dosage increases. In contrast, the degree of protection rose when dosages of the booster immunizations were increased over a 25-fold range. Four weekly peroral, but only two biweekly parenteral, booster immunizations were necessary to achieve strong protection; biweekly combined parenteral and peroral booster immunizations yielded both strong, immediate and extended protection. The degree of protection against the toxin correlated with that against viable bacteria and with elevated serum antitoxin titers: all seven groups with a protection index of greater than 5 against the toxin had strong protection against heat-labile toxin-producing strains and fourfold or greater increases in the antitoxin titers, whereas none of the nine groups with a protection index of less than 3 was protected against bacteria or had an equivalent antitoxin response. These observations show that once an adequate parenteral primary immunization is given, the degree of protection is influenced primarily by the dosage of the booster immunizations, the necessary number of which is dependent on their route of administration.