To develop an animal model of Kaposi sarcoma–associated herpesvirus (KSHV) infection uniquely suited to evaluate longitudinal patterns of viral gene expression, cell tropism, and immune responses, we injected NOD/SCID mice intravenously with purified virus and measured latent and lytic viral transcripts in distal organs over the subsequent 4 months. We observed sequential escalation of first latent and then lytic KSHV gene expression coupled with electron micrographic evidence of virion production within the murine spleen. Using novel technology that integrates flow cytometry with immunofluorescence microscopy, we found that the virus establishes infection in murine B cells, macrophages, NK cells, and, to a lesser extent, dendritic cells. To investigate the potential for human KSHV–specific immune responses within this immunocompromised host, we implanted NOD/SCID mice with functional human hematopoietic tissue grafts (NOD/SCID-hu mice) and observed that a subset of animals produced human KSHV–specific antibodies. Furthermore, treatment of these chimeric mice with ganciclovir at the time of inoculation led to prolonged but reversible suppression of KSHV DNA and RNA levels, suggesting that KSHV can establish latent infection in vivo despite ongoing suppression of lytic replication.