The location of cis-acting regulatory regions within the long terminal repeat (LTR) of the human T-cell leukemia virus type I (HTLV-1) was determined. The sequences present between nucleotides -350 and -55 (cap site +1) contain an enhancer element that is active in lymphoid and nonlymphoid cell lines. The sequences located near the "TATA" and RNA initiation sites contain a promoter, the activity of which can be augmented by homologous and heterologous enhancer elements. A region responsive to trans-acting transcription factors present in HTLV-I- and HTLV type II-infected cells is located between nucleotides -159 and +315. HTLV-I LTR deletion mutants respond in a similar manner both to the trans-acting factors present in infected cells and to the tat protein encoded by the x-lor region of the genome, thus providing further evidence that the tat protein mediates transcriptional trans-activation of the LTR in HTLV-infected cells.