The dynamics of the establishment of, and reactivation from, gammaherpesviruses latency has not been quantitatively analyzed in the natural host. Gammaherpesvirus 68 (γHV68) is a murine gammaherpesvirus genetically related to primate gammaherpesviruses that establishes a latent infection in infected mice. We used limiting dilution reactivation (frequency of cells reactivating γHV68 in vitro) and limiting dilution PCR (frequency of cells carrying γHV68 genome) assays to compare γHV68 latency in normal (C57BL/6) and B-cell-deficient (MuMT) mice. After intraperitoneal (i.p.) inoculation, latent γHV68 was detected in the spleen, bone marrow, and peritoneal cells. Both B-cell-deficient and C57BL/6 mice established latent infection in peritoneal cells after either i.p. or intranasal (i.n.) inoculation. In contrast, establishment of splenic latency was less efficient in B-cell-deficient than in C57BL/6 mice after i.n. inoculation. Analysis of reactivation efficiency (reactivation frequency compared to frequency of cells carrying γHV68 genome) revealed that (i) regardless of route or mouse strain, splenic cells reactivated γHV68 less efficiently than peritoneal cells, (ii) the frequency of cells carrying γHV68 genome was generally comparable over the course of infection between C57BL/6 and B-cell-deficient mice, (iii) between 28 and 250 days after infection, cells from B-cell-deficient mice reactivated γHV68 10- to 100-fold more efficiently than cells from C57BL/6 mice, (iv) at 7 weeks postinfection, B-cell-deficient mice had more genome-positive peritoneal cells than C57BL/6 mice, and (v) mixing cells (ratio of 3 to 1) that reactivated inefficiently with cells that reactivated efficiently did not significantly decrease reactivation efficiency. Consistent with a failure to normally regulate chronic γHV68 infection, the majority of infected B-cell-deficient mice died between 100 and 200 days postinfection. We conclude that (i) B cells are not required for establishment of γHV68 latency, (ii) there are organ-specific differences in the efficiency of γHV68 reactivation, (iii) B cells play a crucial role in regulating reactivation of γHV68 from latency, and (iv) B cells are important for controlling chronic γHV68 infection.