Mammalian histone gene transcription is increased approximately fivefold during the transition from the G1 phase to the S phase of the cell cycle. In this study, we present a detailed in vivo analysis of the human histone H2b promoter, which establishes that transcriptional regulation of this gene is mediated by a subtype-specific consensus element containing the core octanucleotide ATTTGCAT. Our results demonstrate that the activity of this sequence is specific for S phase. Comparative analysis of different replication variant mammalian histone gene promoters and our knowledge of the transcription factors interacting with the human histone H2b and H4 promoters allow us to conclude that coordinate regulation of histone gene transcription in higher eukaryotes is mediated by distinct factors. We propose a simple model for transcriptional regulation of mammalian histone gene expression, which incorporates both the distinct features of the individual histone gene promoters and the apparent functional equivalence of the specific sequence elements regulating transcription of each histone gene subtype.