Transcriptional activation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter element is regulated by the essential viral Tat protein that binds to the viral TAR RNA target and recruits a positive transcription elongation complex (P-TEFb). We have used a stepwise transcription approach and a highly sensitive assay to determine the dynamics of interactions between HIV-1 Tat and the transcription complexes actively engaged in elongation. Our results demonstrate that Tat protein associates with RNA polymerase II complexes during early transcription elongation after the promoter clearance and before the synthesis of full-length TAR RNA transcript. This interaction of Tat with RNA polymerase II elongation complexes is P-TEFb-independent. Our results also show that there are two Tat binding sites on each transcription elongation complex; one is located on TAR RNA and the other one on RNA polymerase II near the exit site for nascent mRNA transcripts. These findings suggest that two Tat molecules are involved in performing various functions during a single round of HIV-1 mRNA synthesis.