Lentiviruses translate their structural and enzymatic proteins from unspliced and singly spliced viral mRNAs (vRNAs). Export to the cytoplasm of the incompletely spliced transcripts is mediated by the viral Rev protein. Rev binds to a highly structured region of the unspliced/singly spliced RNA, termed the Rev responsive element (RRE), multimerizes, and tethers the RNA to cellular export factors to cross the nuclear membrane. While Rev-RRE mediated nuclear export is well studied in human immunodeficiency virus type 1 (HIV-1), the interaction between the equine infectious anemia virus (EIAV) Rev and the RRE is less well studied. Chemical footprinting analysis indicates Rev interacts with two regions on the RNA, termed Rev binding region one (RBR-1) and Rev binding region 2 (RBR-2). RBR-1 contains a 56nt sequence termed the minRRE sufficient to both bind Rev and support nuclear export. The minRRE spans Rev exon 1 and an exon splicing enhancer (ESE) necessary for Rev exon 1 splicing. RBR-2 was previously functionally uncharacterized. RBR-2 contains predicted structural motifs found in several other lentiviruses, and is required for Rev high affinity binding, suggesting this region is biologically relevant and may be involved in Rev nuclear export. Here we examined the effect of deleting RBR-2 on nuclear export, virus structural gene expression, and virion production, and Rev-SF2/ASF competition. Our findings indicate RBR-2 is neither necessary nor sufficient for nuclear export, and deletion of RBR-2 does not affect either virus structural gene expression or virion production, suggesting RBR-1 is the primary binding site and RRE for Rev. The RBR-1 sequence secondary structure and the genomic intronic location is distinct from that of HIV-1 and other lentivirus RREs. It is still unclear if RBR-2 has a functional role in virus replication.