The Rex protein of the human T-cell leukemia virus type II (HTLV-II), Rex-II, plays a central role in regulating the expression of the structural genes of this retrovirus. Rex-II acts posttranscriptionally by inducing the cytoplasmic expression of the incompletely spliced viral mRNAs that encode the Gag and Env structural proteins and the enzymes derived from the pol gene. We now define a 295-nucleotide cis-acting regulatory element within the 3' long terminal repeat of HTLV-II that is required for the effects of Rex-II. This Rex-II response element (RexIIRE) corresponds to a predicted, highly stable RNA secondary structure and functions when present in the sense but not in the antisense orientation. The RexIIRE confers responsiveness not only to Rex-II but also to the Rex protein of HTLV-I. Deletion and substitution mutagenesis of the RexIIRE permitted identification of a small subregion within the larger element critically required for Rex-II responsiveness and further suggested that the structurally distinct RexIIREs generated from the 5' and 3' long terminal repeats of HTLV-II may differentially regulate the cytoplasmic expression of unspliced gag-pol and singly spliced env mRNAs. While the Rev protein of human immunodeficiency virus type 1 fails to function via the RexIIRE, the Rex-II protein, like Rex-I, can functionally replace the Rev protein of human immunodeficiency virus type 1 via its interaction with the Rev response element (RevRE).