During primary rotavirus (RV) infection, CD8+ T cells play an important role in viral clearance as well as providing partial protection against reinfection. CD4+ T cells are essential for maximal development of RV-specific intestinal immunoglobulin A. In this study, we took advantage of the cytokine flow cytometry technique to obtain a detailed map of H-2b- and H-2d-restricted CD8+ and CD4+ T-cell epitopes from the RV proteins VP6 and VP7. Three new CD8+ T-cell epitopes (H-2d and H-2b restricted) and one new CD4+ T-cell epitope (H-2d and H-2b restricted) were identified. Using these newly identified targets, we characterized the development and specificity of cellular immune responses in C57BL/6 and BALB/c mice during acute infection of infants and adults. We found that both the CD4+ and CD8+ responses peaked on days 5 to 7 after infection and then declined rapidly. Interestingly, both the response kinetics and tissue distributions were different when epitopes on VP6 and VP7 were compared. VP6 elicited a response which predominated in the intestine, while the response to VP7 was more systemic. Additionally, the T-cell responses elicited after homologous versus heterologous infection differed substantially. We found that during homologous infection, there was a greater response toward VP6 than that toward VP7, especially in the intestine, while after heterologous infection, this was not the case. Finally, in suckling mice, we found two peaks in the CD8 response on days 7 and 14 postinfection, which differed from the single peak found in adults and likely mimics the biphasic pattern of rotavirus shedding in infant mice.