Feline immunodeficiency virus (FIV) is a naturally occurring pathogen that causes an AIDS-like syndrome in domestic cats and is a valuable model system by which criteria for antiviral vaccines and drugs development can be tested. The cell-entry step of the lentivirus life cycle is regarded as a promising target for the development of new generation inhibitors. We have previously described potent in vitro anti-FIV activity associated with a synthetic octapeptide, termed C8 (Ac-Trp-Glu-Asp-Trp-Val-Gly-Trp-Ile-NH2), containing the Trp-rich motif of FIV transmembrane glycoprotein, which shares a common structural framework with the corresponding molecule of HIV and appears to play a similar role in cell entry. In this report, in an attempt to develop simpler potential fusion inhibitors to be tested in vivo, we describe further studies focused on synthetic peptide analogues of C8. Since C8 inhibitory activity is dependent upon the Trp motif, we systematically replaced these residues with bulky and/or aromatic natural and unnatural amino acids, in order to develop a rational structure-activity relationship. Furthermore, the amino acids located between the Trp residues, which are not crucial for inhibitory activity, were replaced by simple alkyl spacers of appropriate length. Design, NMR structural analysis, in vitro anti-FIV activity in lymphoid cell cultures, and serum stability of these new analogues are reported. The final results indicate that a simpler hexapeptide (Ac-Nal2-Ape-Nal2-Ape-Nal2-Ile-NH2; Nal2 = 3-naphthalen-2-yl-L-alanine, Ape = 5-aminopentanoic acid), almost entirely made up of unnatural amino acid residues, has markedly increased enzymatic stability, while maintaining strong antiviral potency in vitro.