Feline immunodeficiency virus (FIV) is a lentivirus inducing an AIDS-like disease in cats, thus providing an interesting model for AIDS study. FIV and HIV-1 possess a similar genomic arrangement of structural and non-structural genes, whose expression is regulated by related genetic mechanisms. On the DNA strand complementary to the HIV-1 envelope messenger, an open reading frame (ORF) has been identified which encompasses the Rev Responsive Element (RRE), a cis-acting element critical to HIV gene expression. This ORF was highly conserved among HIV-1 isolates and had the potential to encode a hydrophobic protein. A corresponding antisense transcript was detected in cells infected with HIV-1, and appeared to encode an antisense protein. In the present study, we have identified a well-conserved antisense ORF, also coincident with the RRE region, in the genome of 21 FIV isolates. The predicted protein, 103 amino acids in length, is highly hydrophobic, as is the case for that of HIV-1. In addition, we have shown that a corresponding transcript, complementary to the transmembrane sequence of the FIV envelope gene, was produced in different cellular and viral contexts, that is, ex vivo and in vivo in FIV infected cell lines and tissues of infected cats, respectively. Expression of antisense transcripts might therefore be of general importance in the natural history of retroviruses.