Expression of the incompletely spliced HTLV-I mRNAs relies on the viral posttranscriptional activator Rex, whose interaction with the Rex-responsive element (RXRE) overcomes effects of cis-acting repressive sequences (CRSs). Studies based on heterologous reporter plasmids identified an intronic CRS in the 5' LTR and a CRS that overlaps with the RXRE. The present study investigated the effects of these elements in the context of spliced viral mRNAs encoding p21Rex (mRNA 1-3), Tax/Rex (mRNA 1-2-3), and Tof (mRNA 1-2-B). All three mRNAs were inefficiently expressed when transcribed in their mature intronless form, with the p21Rex mRNA showing the weakest expression. In contrast, efficient expression of p21Rex was obtained from a plasmid containing the 5' LTR and 3' portion of the genome that encoded a spliceable RNA. The defective expression of the intronless mRNAs reflected the inhibitory activity of the RXRE and the lack of 5' intronic sequences. Insertion of an intronic 5' LTR segment located upstream of the 5' CRS overcame Rex dependence conferred by the RXRE. The activity of this segment was mapped to the major splice donor and sequences overlapping with, but functionally distinct from, a previously described transcriptional enhancer. The three mRNAs responded differently to Rex and to insertion of the constitutive transport element of simian retrovirus type 1. Taken together, these results suggest that expression of the spliced mRNAs is controlled by the relative influence of positive and negative sequences present on the primary transcript as well as the Rex-RXRE interaction.