Histone gene expression is cell cycle regulated at the transcriptional and the post-transcriptional levels. Upon entry into S phase, histone gene transcription is stimulated 2- to 5-fold and peaks within 1-3 hr of the initiation of DNA synthesis. We have delineated the proximal promoter element responsible for cell cycle-dependent transcription of a human histone H4 gene in vivo. Our results indicate that H4 cell cycle-dependent transcriptional regulation is mediated by an 11-base-pair element, the cell cycle element (5'-CTTTCG-GTTTT-3'), that resides in the in vivo protein-DNA interaction site, site II (nucleotides -64 to -24). The H4 cell cycle element functions as a master switch for expression of the FO108 human histone H4 gene in vivo; mutations within the H4 cell cycle element drastically reduce the level of expression as well as abrogate cell cycle-regulated transcription. Furthermore, these mutations result in a loss of binding in vitro of the cognate nuclear factor HiNF-M. In vivo competition analysis indicates that the cell cycle element mediates specific competition for a DNA-binding factor, presumably HiNF-M, that is a rate-limiting step in transcription of this H4 gene.