We studied regulation of human immunodeficiency virus-1 (HIV-1) transcription by Tat and, for comparative purposes, by the adenovirus E1A protein. These two trans-activators exerted different effects. Two classes of HIV-1-promoted cytoplasmic RNA were detected, one class corresponding to full-length transcripts and the other to transcripts ending 55 and 59 nucleotides from the transcription start. Tat increased the level of the full-length class only, whereas E1A increased the levels of both classes of RNA. We also measured the effects of Tat and E1A on RNA synthesis rates. Without trans-activators, HIV-1-directed transcription was relatively weak and exhibited a marked polarity. Both Tat and E1A dramatically increased promoter-proximal transcription, while only Tat suppressed transcriptional polarity. Mutations in the TAR element did not influence basal transcription rates or the response to E1A, but eliminated trans-activation by Tat. We propose that Tat acts through TAR to increase initiation complex formation on the HIV-1 promoter and to stabilize complexes during elongation.