The objective of this study was to identify cellular and organ targets of acute feline immunodeficiency virus (FIV) infection in vivo. Tissues of FIV-infected cats were studied at eight time points during the first 3 months after experimental infection. FIV nucleic acids were first detected by in situ hybridization 21 days after infection, approximately 1.5 weeks after lymph node enlargement was first observed and 3 weeks before the primary acute flu-like illness. The majority of FIV-infected cells were present in lymphoid organs, though low numbers of infected cells were noted in nonlymphoid organs as well. Germinal centers harbored many of the FIV-infected cells within lymphoid tissues. The thymic cortex was also a major site of early infection. Combined in situ hybridization and immunohistochemistry revealed that T lymphocytes were the primary target of early FIV infection in tissues of cats before the onset of clinical signs of acute illness. An unidentified population of mononuclear cells and a few macrophages were also infected. During the ensuing acute flu-like illness, the proportion of FIV-infected macrophages in tissues increased dramatically. This early shift in the predominant cellular localization of FIV from T lymphocytes to macrophages may be important for establishing viral persistence.