Maximal transcription of a prototypical cell cycle controlled histone H4 gene requires a proliferation-specific in vivo genomic protein/DNA interaction element, Site II. Three sequence-specific transcription factors interact with overlapping recognition motifs within Site II: interferon regulatory factor IRF-2 (HiNF-M), the putative H4 subtype-specific protein H4TF-2 (HiNF-P), and HiNF-D which represents a complex of the homeodomain protein CDP/cut, CDC2, cyclin A and pRB. However, natural sequence variation in the Site II sequences of different human H4 genes abolishes binding of specific trans-acting factors; the functional consequences of these variations have not been investigated. To address the precise contribution of H4 promoter factors to the level of H4 gene transcription, we performed a systematic mutational analysis of Site II transcriptional motifs. These mutants were tested for ability to bind each of the Site II cognate proteins, and subsequently evaluated for ability to confer H4 transcriptional activity using chimeric H4 promoter/CAT fusion constructs in different cell types. We also analyzed the effect of over-expressing IRF-2 on CAT reporter gene expression driven by mutant H4 promoters and assessed H4 transcriptional control in cells nullizygous for IRF-1 and IRF-2. Our results show that the recognition sequence for IRF-2 (HiNF-M) is the dominant component of Site II and modulates H4 gene transcription levels by 3 fold. However, the overlapping recognition sequences for IRF-2 (HiNF-M), H4TF-2 (HiNF-P) and CDP/cut (HiNF-D) together modulate H4 gene transcription levels by at least an order of magnitude. Thus, maximal activation of H4 gene transcription during the cell cycle in vivo requires the integrated activities of multiple transcription factors at Site II. We postulate that the composite organization of Site II supports responsiveness to multiple signalling pathways modulating the activities of H4 gene transcription factors during the cell cycle. Variations in Site II sequences among different H4 genes may accommodate differential regulation of H4 gene expression in cells and tissues with unique phenotypic properties.