Immunization with naked plasmid DNA effectively induces both humoral and cell-mediated immunity to vaccine antigens and can confer protection against numerous infectious diseases. To explore the potential use of DNA immunization to induce rotavirus-specific immune responses, we used plasmid DNA encoding the VP4 gene of bovine rotavirus (BRV). Intrasmuscular injection of the plasmid encoding the VP4 gene into C57BI/6 mice induced cell-mediated immunity as measured by cytokine production. Although DNA immunization did not induce a detectable BRV-specific antibody response, DNA-immunized animals were primed for antibody production and a cellular immune response. Following viral inoculation, the immunized animals displayed an enhanced number of BRV-specific antibody-secreting cells and cytotoxic activity. The immune response induced by DNA immunization alone or followed by viral inoculation was biased toward IFN-gamma production (Th1-like). CD4+ lymphocytes were the major source of IFN-gamma production in the spleen following DNA immunization. In contrast, a balanced cytokine production was observed in the spleens of animals receiving whole virus. These experiments showed that DNA immunization with a gene encoding the VP4 protein of BRV stimulated a Th1-like immune response in mice, and this bias in the immune response persisted following exposures to whole virus.