The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1), gp160, is synthesized as a protein precursor that when proteolytically cleaved yields two subunits, gp120 and gp41. gp120 is the surface glycoprotein on HIV-1 responsible for binding to CD4, and gp41 is the transmembrane glycoprotein involved in the membrane fusion process. gp41 is divided into the N-terminal fusion peptide, the heptad repeat 1 (HR1) and HR2 regions, and the C-terminal transmembrane region, which are collectively responsible for virus fusion and entry into the cell. Synthetic peptides derived from the HR2 and HR1 regions of HIV-1(LAI) have been shown to prevent virus-cell fusion and infection in vitro. In phase II clinical trials in HIV patients, data revealed that T20 has antiviral efficacy and is well tolerated. Similar results were obtained in vitro with HIV-2 and simian immunodeficiency virus, supporting the conservation of the gp41 ectodomain among lentiviruses. Feline immunodeficiency virus (FIV) infection in the cat has been used as a model to develop potential antivirals for HIV. To determine if synthetic gp40 analogs capable of inhibiting FIV infection could be identified, 15 overlapping 35-amino-acid peptides derived from the C-terminal HR2 domain of FIV gp40 were synthesized. These peptides were tested for efficacy against FIV in a syncytium-forming assay with FIV-infected CrFK cells and HeLa cells expressing the FIV receptor CXCR4. Several peptides exhibited activity at the nanogram level. Antiviral activity was confirmed by suppression of reverse transcriptase in a FIV feline CD4(+)-T-cell (FCD4-E) acute-infection assay. These data demonstrate that synthetic peptides derived from the HR2 domain of the FIV gp41 protein are effective inhibitors of FIV infection.