Studies on functional compatibility of various Rev proteins derived from all known human and simian immunodeficiency virus subgroups have shown that this essential gene product is not always exchangeable among the viruses. In an attempt to map the region of Rev proteins responsible for the observed nonreciprocal complementation, hybrid genomic Rev expression vectors were constructed by exchanging the first and second exons of rev genes, and were examined for their abilities to activate reporter clones by transfection. With one exception, the second coding exon of rev gene determined the functional specificity of Rev proteins.