Feline immunodeficiency virus (FIV) is a naturally occurring T-cell tropic lentiviral disease of felids with many similarities to HIV/AIDS in humans. Similar to primate lentiviral-host interactions, feline APOBEC3 (A3) has been shown to inhibit FIV infection in a host-specific manner and feline A3 degradation is mediated by FIV Vif. Further, infection of felids with non-native FIV strains results in restricted viral replication in both experimental and naturally occurring infections. However, the link between molecular A3-Vif interactions and A3 biological activity during FIV infection has not been well characterized. We thus examined expression of the feline A3 genes A3Z2, A3Z3 and A3Z2-Z3 during experimental infection of domestic cats with host-adapted domestic cat FIV (referred to as FIV) and non-adapted Puma concolor FIV (referred to as puma lentivirus, PLV). We determined A3 expression in different tissues and blood cells from uninfected, FIV-infected, PLV-infected and FIV/PLV co-infected cats; and in purified blood cell subpopulations from FIV-infected and uninfected cats. Additionally, we evaluated regulation of A3 expression by cytokines, mitogens, and FIV infection in cultured cells. In all feline cells and tissues studied, there was a striking difference in expression between the A3 genes which encode FIV inhibitors, with A3Z3 mRNA abundance exceeding that of A3Z2-Z3 by 300-fold or more. Interferon-alpha treatment of cat T cells resulted in upregulation of A3 expression, while treatment with interferon-gamma enhanced expression in cat cell lines. In cats, secondary lymphoid organs and peripheral blood mononuclear cells (PBMC) had the highest basal A3 expression levels and A3 genes were differentially expressed among blood T cells, B cells, and monocytes. Acute FIV and PLV infection of cats, and FIV infection of primary PBMC resulted in no detectable change in A3 expression with the exception of significantly elevated A3 expression in the thymus, the site of highest FIV replication. We conclude that cat A3 expression is regulated by cytokine treatment but, by and large, lentiviral infection did not appear to alter expression. Differences in A3 expression in different blood cell subsets did not appear to impact FIV viral replication kinetics within these cells. Furthermore, the relative abundance of A3Z3 mRNA compared to A3Z2-Z3 suggests that A3Z3 may be the major active anti-lentiviral APOBEC3 gene product in domestic cats.