Chromatin organization of eukaryotic promoters is increasingly recognized as an important factor in the regulation of transcription in vivo. To determine the role of chromatin in HIV‐1 expression, we have examined the nucleosome organization of the promoter of HIV‐1 under low and high transcription rates. Independently of the cell line examined, nucleosomes are precisely positioned in the viral 5′ long terminal repeat (5′ LTR) and define two large nucleosome‐free regions encompassing nt 200–450 and 610–720. A nucleosome positioned between these two regions, immediately after the transcription initiation site (nuc‐1), is disrupted following TPA or TNF‐alpha treatment. The disruption of nuc‐1 from DNA is independent of DNA replication since it is completed in 20 min and independent of transcription as it is alpha‐amanitin insensitive. A model is proposed in which nuc‐1 plays an organizing role in the HIV‐1 promoter to bring in close proximity factors bound to DNA in the two nucleosome‐free regions, upstream and downstream of the site of transcription initiation. These results define chromatin as an integral component of the HIV‐1 transcriptional regulatory machinery and identify a chromatin transition associated with activation of viral gene expression.