Tat expression is required for efficient human immunodeficiency virus type 1 (HIV-1) reverse transcription. In the present study, we generated a series of 293 cell lines that contained a provirus with a tat gene deletion (Deltatat). Cell lines that contained Deltatat and stably transfected vectors containing either wild-type tat or a number of tat mutants were obtained so that the abilities of these tat genes to stimulate HIV-1 gene expression and reverse transcription could be compared. tat genes with mutations in the amino terminus did not stimulate either viral gene expression or HIV-1 reverse transcription. In contrast, tat mutants in the activation, core, and basic domains of Tat did not stimulate HIV-1 gene expression but markedly stimulated HIV-1 reverse transcription. No differences in the levels of virion genomic RNA or tRNA3Lys were seen in the HIV-1 Deltatat viruses complemented with either mutant or wild-type tat. Finally, overexpression of the Tat-associated kinases CDK7 and CDK9, which are involved in Tat activation of HIV-1 transcription, was not able to complement the reverse transcription defects associated with the lack of a functional tat gene. These results indicate that the mechanism by which tat modulates HIV-1 reverse transcription is distinct from its ability to activate HIV-1 gene expression.