Feline immunodeficiency virus (FIV) causes an AIDS-like syndrome in the domestic cat. As many parallels exist in both the pathology and molecular structure of FIV and HIV, the cat is a valuable small animal model for the development of broad-based treatment for lentivirus infections. HIV and FIV primarily target CD4+ T-cells, but HIV uses CD4 as a primary binding receptor, whereas FIV utilizes CD134. After interaction with the primary binding receptor, however, both FIV and T-cell tropic HIV-1 use the chemokine receptor CXCR4 for cell entry. Identification of structures critical for receptor binding by FIV isolates will thus contribute to defining potential targets for intervention. In this study, we employed site-directed mutagenesis to investigate the importance of specific residues on the V3 loop of the FIV envelope for CXCR4 binding. We identified three basic residues and one conserved residue as critical for CXCR4 binding, but not for interactions with CD134. Furthermore, receptor binding abilities correlated with entry efficiencies. Our findings provide insights into FIV/receptor interactions in the entry process and may prove useful in the development of novel host targets for broad-based therapies.