Feline immunodeficiency virus (FIV) is a lentivirus which infects domestic cats, causing an acquired immunodeficiency syndrome (AIDS). The aim of the present work was the development of an immunoassay for the diagnosis of FIV infection, using synthetic peptides from FIV envelope (Env) glycoproteins. Four peptides (8 to 11 amino acids long) corresponding to group-specific epitopes of FIV Env extracellular (SU) or transmembrane (TM) glycoproteins were synthesized. They were evaluated by enzyme-linked immunosorbent assay (ELISA) for immunoreactivity with sera from naturally or experimentally FIV-infected cats. One of these, P237, corresponds to a conserved nonapeptide of FIV TM, folded as a loop between two cysteines. ELISA performed with P237 on 171 sera from FIV-infected cats and 46 sera from specific-pathogen-free cats showed no false positive cases and 100% detection of infected cat sera. Moreover, 47 pet cat sera which were negative with a whole virus-based-ELISA were tested with the P237 ELISA: 2 out of 47 showed reactivity. FIV infection of these two cats was confirmed by radio-immunoprecipitation assay. Temporal studies performed on serial serum samples from experimentally infected cats detected antibodies to P237 three to five weeks after inoculation of virus. Thus, the P237 ELISA is a sensitive and specific immunoassay for early detection of antibodies to FIV. In addition, this synthetic nonapeptide is easier to produce and purify than virus preparations or recombinant proteins.