Serum and intestinal humoral immune responses to rotavirus proteins VP2, VP4, VP6, VP7, NSP2, and NSP4 were quantitatively compared in mice infected with a homologous murine rotavirus (EHPw) or a heterologous simian rotavirus (RRV). Viral protein-specific antibody responses were measured by an immunohistochemistry assay that uses recombinant baculovirus-expressed rotavirus proteins as antigens. In serum, IgG responses to VP6 were dominant and comparable in both RRV- and EHPw-infected groups, but responses to VP2, VP4, VP7, and NSP2 were higher in RRV infection. In feces, IgA responses to VP2, VP4, and VP6 were higher in EHPw-infected mice, but responses to VP7 and NSP2 were detected only in the RRV-infected group. These findings indicate that immune responses to homologous and heterologous rotavirus infection vary both quantitatively and qualitatively. Differences in humoral responses may play a role in the differences in protection induced following homologous or heterologous rotavirus infection.