Spliced messages encoded by two distinct strains of feline immunodeficiency virus (FIV) were identified. Two of the cDNA clones represented mRNAs with bicistronic capacity. The first coding exon contained a short open reading frame (orf) of unknown function, designated orf 2. After a translational stop, this exon contained the L region of the env orf. The L region resides 5' to the predicted leader sequence of env. The second coding exon contained the H orf, which began 3' to env and extended into the U3 region of the long terminal repeat. The in-frame splicing of the L and H orfs created the FIV rev gene. Site-directed antibodies to the L orf recognized a 23-kDa protein in infected cells. Immunofluorescence studies localized Rev to the nucleoli of infected cells. The Rev-responsive element (RRE) of FIV was initially identified by computer analysis. Three independent isolates of FIV were searched in their entirety for regions with unusual RNA-folding properties. An unusual RNA-folding region was not found at the Su-TM junction but instead was located at the end of env. Minimal-energy foldings of this region revealed a structure that was highly conserved among the three isolates. Transient expression assays demonstrated that both the Rev and RRE components of FIV were necessary for efficient reporter gene expression. Cells stably transfected with rev-deleted proviruses produced virion-associated reverse transcriptase activity only when FIV Rev was supplied in trans. Thus, FIV is dependent on a fully functional Rev protein and an RRE for productive infection.