The human T-cell leukemia virus (HTLV-1) Rex protein is essential for the cytoplasmic accumulation of incompletely spliced transcripts that code for the viral structural proteins. In this study effects of Rex on the amounts of total, spliced and unspliced RNA from HTLV-1 were determined. In transfected fibroblasts Rex production resulted in reduced amounts of spliced RNA and increased quantities of unspliced RNA in the nucleus. However, the total amount of viral RNA was not affected and the stability of spliced transcripts was not changed, thus indicating that only the rate of splicing was reduced. Rex action also reduced splicing in immortalized human cord blood T-cells. However, the total amount of viral transcripts and the stability of unspliced RNA in these cells were also increased in the presence of Rex. This indicates that Rex also prevents the degradation of unspliced transcripts in T-cells. The changes in the relative amounts of spliced and unspliced RNA induced by Rex were observed not only in the cytoplasm but also in the nucleus. Thus Rex affects the nucleocytoplasmic transport, splicing and stability of HTLV-1 RNA in the nucleus. These observations may suggest that Rex directs the unspliced viral RNA to the cytoplasm via a nuclear compartment that is not accessible to splicing and degradation factors.