Sequence analysis of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) does not reveal a polyadenylation consensus sequence, AAUAAA, close to the polyadenylation site at the 3' end of the viral RNA. Using site-directed mutagenesis, we demonstrated that two cis-acting signals are required for efficient RNA processing in HTLV-I LTR: (i) a remote AAUAAA hexamer at a distance of 276 nucleotides upstream of the polyadenylation site, and (ii) the 20-nucleotide GU-rich sequence immediately downstream from the poly(A) site. It has been postulated that the folding of RNA into a secondary structure juxtaposes the AAUAAA sequence, in a noncontiguous manner, to within 14 nucleotides of the polyadenylation site. To test this hypothesis, we introduced deletions and point mutations within the U3 and R regions of the LTR. RNA 3'-end processing occurred efficiently at the authentic HTLV-I poly(A) site after deletion of the sequences predicted to form the secondary structure. Thus, the genetic analysis supports the hypothesis that folding of the HTLV-I RNA in the U3 and R regions juxtaposes the AAUAAA sequence and the poly(A) site to the correct functional distance. This unique arrangement of RNA-processing signals is also found in the related retroviruses HTLV-II and bovine leukemia virus.