Expression of many histone H4 genes is stringently controlled during the cell cycle to maintain a functional coupling of histone biosynthesis with DNA replication. The histone H4 multigene family provides a paradigm for understanding cell cycle control of gene transcription. All functional histone H4 gene copies are highly conserved in the mRNA coding region. However, the putative promoter regions of these H4 genes are divergent. We analyzed three representative mouse H4 genes to assess whether variation in H4 promoter sequences has functional consequences for the relative level and temporal control of expression of distinct H4 genes. Using S1 nuclease protection assays with gene-specific probes and RNA from synchronized cells, we show that the mRNA level of each H4 gene is temporally coupled to DNA synthesis. However, there are differences in the relative mRNA levels of these three H4 gene copies in several cell types. Based on gel shift assays, nucleotide variations in the promoters of these H4 genes preclude or reduce binding of several histone gene transcription factors, including IRF2, HiNF-D, SP-1 and/or YY1. Therefore, differential regulation of H4 genes is directly attributable to evolutionary divergence in H4 promoter organization which dictates the potential for regulatory interactions with cognate H4 transcription factors. This regulatory flexibility in H4 promoter organization may maximize options for transcriptional control of histone H4 gene expression in response to the onset of DNA synthesis and cell cycle progression in a broad spectrum of cell types and developmental stages.