The Rex protein of human T-cell leukemia virus type I (HTLV-I) was expressed in bacteria and partially purified. Rex was shown to bind in vitro specifically to an RNA sequence located in the 3' long terminal repeat of HTLV-I, named Rex-responsive element (RXRE). Rex also bound in vitro to the human immunodeficiency virus type 1 (HIV-1) Rev-responsive element (RRE), while purified HIV-1 Rev protein did not bind to the RXRE. The binding results obtained in vitro are therefore in agreement with the nonreciprocal function of Rev and Rex in vivo. Rex binds specifically to both RRE and RXRE and activates expression in both HIV-1 and HTLV-I, while Rev binds to RRE and activates only HIV-1. Binding of Rex to RRE deletion mutants previously shown to lack either the Rev-responsive or the Rex-responsive portion suggested preferential binding of Rex to a distinct target within the RRE. These results demonstrated that Rex, like Rev, acts by binding to a specific RNA target.