The replication of human immunodeficiency virus type 1 (HIV-1) can be inhibited by noncytolytic CD8(+) T cell mediated suppression, an immune response that specifically targets HIV-1 gene expression. Clinical studies demonstrate that this immune response may play an important role in the host defense against HIV infection. In this study, we examined the distinct steps in viral gene expression for inhibition by noncytolytic CD8(+) T cells. A primary HIV-1 infection system of CD4(+) enriched peripheral blood mononuclear cells was utilized to examine the HIV-1 life cycle as a relevant ex vivo system. Established CD8(+) T cell lines from two HIV(+) long-term nonprogressors were used to examine differences at the level of transcriptional initiation and elongation of the HIV genome. This infection system coupled with the results from real-time measurement of newly transcribed RNA transcripts determined that there was a significant decrease (5-8 fold) in short intracellular viral RNA transcripts. These data strongly favor a role for the initiation of virus transcription in noncytolytic CD8(+) T cell mediated suppression.