The architecture of a retroviral genome presents some unusual features for transcriptional regulation because of duplication of the transcriptional control sequences in the 5' and 3' long terminal repeats (LTRs). We have studied the transcriptional activity of the 5' and 3' LTRs of human immunodeficiency virus type 1 (HIV-1) vectors. Using full-length HIV molecular clones, we demonstrate that both LTRs function as Tat-inducible promoters. However, the absolute levels of transcription were found to be much higher for the 5' LTR than for the 3' LTR promoter. When transcription was assayed for an integrated HIV-1 provirus, we also found that the upstream 5' LTR element was the major transcriptional promoter. 3' LTR transcription, however, can be triggered by inactivation of the 5' LTR promoter. Likewise, 5' LTR transcription is induced in constructs lacking a functional 3' LTR promoter. This phenomenon of promoter suppression may have important implications for the design of HIV-based retrovirus vectors.