In this study we used nuclear extracts from centrifugally elutriated cell populations to study histone H1 transcriptional regulation during the cell cycle. Analysis of mutations within the H1 promoter establish that both of the H1 subtype-specific consensus elements participate in induction of transcription upon entry into S phase. The DNA binding activity of H1TF2, which specifically interacts with the H1 proximal subtype-specific element, is increased in S-phase nuclear extracts, whereas no increase in DNA binding is observed for the H1 distal subtype-specific DNA transcription factor H1TF1 or the H2b subtype-specific factor OTF1. These data strongly support the idea that histone gene subtype-specific transcription factors are important for S-phase-dependent expression of histone genes. Further studies of these factors will be important for increased understanding of the transition from G1 to S phase of the mammalian cell cycle.