The Rex protein of human T-cell leukemia viruses (HTLV) is a trans-acting regulator inducing the expression of gag and env mRNA containing the introns. The rex gene can also induce expression of unspliced RNA of human immunodeficiency viruses (HIV). We have analyzed the level of spliced and unspliced RNAs in nucleus and cytoplasm to understand the mechanism by which the Rex protein modulates RNA processing. With the gag gene of HTLV-1, the unspliced RNA was accumulated by Rex protein in both nucleus and cytoplasm. However, the apparent effects on nuclear unspliced RNA depended on the reporter genes: with the env gene of HTLV-1 as well as that of HIV-1, Rex did not accumulate the unspliced RNA in nucleus, but did so only in cytoplasm. These results clearly indicate that Rex protein not only activates the nuclear export of unspliced RNA, but also modulates some steps of RNA processing before the splicing, probably through stabilization of the precursor RNA.