The human immunodeficiency virus type 1 (HIV-1) Tat protein is a transcriptional activator that is essential for efficient viral gene expression and replication. Tat increases the level of full-length transcripts from the HIV-1 promoter by dramatically enhancing the elongation efficiency of the RNA polymerase II complexes assembled on this promoter. Tat could potentially activate the transcription machinery during initiation, elongation, or both. We used an immobilized HIV-1 promoter template with a reversible lac repressor (LacR) elongation block inserted downstream to dissect the stages in transcription affected by Tat. Transcription complexes assembled in the absence of Tat and blocked by LacR cannot be activated by incubation with Tat alone. These complexes can, however, be activated if Tat is added in combination with cellular factors. In this system, Tat also promoted the assembly of preinitiation complexes capable of elongating efficiently, suggesting that Tat can associate with transcription complex at an early stage. These data indicate that Tat can activate elongation of RNA polymerase by modifying an already elongating transcription complex. The data also suggest the possibility that Tat can interact with initiation complexes.