The relative capacities of intramuscular (im) or oral inoculation of mice with live or inactivated rotavirus to induce a virus-specific humoral immune response in gut-associated lymphoid tissues (GALT) was evaluated. At 4-5 weeks after oral inoculation with live virus, virus-specific IgA was detectable in serum, intestinal contents, and GALT organ culture. Five weeks after im inoculation with live or inactivated virus, virus-specific IgA was detected in GALT organ culture at levels approximately 10-fold less than those found after oral inoculation with live virus. Therefore, neither replication of virus in small intestinal epithelial cells nor presentation of virus at the intestinal mucosal surface was necessary for the induction of virus-specific antibodies by GALT. Possible mechanisms by which im inoculation of virus induces production of virus-specific antibodies by GALT are discussed.