Cytomegalovirus (CMV) is a ubiquitous virus, causing the most common congenital infection in humans, yet a vaccine against this virus is not available. The experimental study of immunity against CMV in animal models of infection, such as the infection of mice with the mouse CMV (MCMV), has relied on systemic intraperitoneal infection protocols, although the infection naturally transmits by mucosal routes via body fluids containing CMV. To characterize the biology of infections by mucosal routes, we have compared the kinetics of virus replication, the latent viral load, and CD8 T cell responses in lymphoid organs upon experimental intranasal and intragastric infection to intraperitoneal infection of two unrelated mouse strains. We have observed that intranasal infection induces robust and persistent virus replication in lungs and salivary glands, but a poor one in the spleen. CD8 T cell responses were somewhat weaker than upon intraperitoneal infection, but showed similar kinetic profiles and phenotypes of antigen-specific cells. On the other hand, intragastric infection resulted in abortive or poor virus replication in all tested organs, and poor T cell responses to the virus, especially at late times after infection. Consistent with the T cell kinetics, the MCMV latent load was high in the lungs, but low in the spleen of intranasally infected mice and lowest in all tested organs upon intragastric infection. In conclusion, we show here that intranasal, but not intragastric infection of mice with MCMV represents a robust model to study short and long-term biology of CMV infection by a mucosal route