The testis-specific H2B histone (TH2B) gene is expressed in pachytene spermatocytes during meiotic prophase I in the absence of any significant DNA synthesis. Unlike somatic histones, synthesis of testis-specific histones is not affected by inhibitors of DNA synthesis. A genomic rat TH2B gene was cloned by using a DNA fragment derived from TH2B cDNA as a probe. Expression of the cloned TH2B was investigated by gene transfer experiments. From these studies, we found that the 5' upstream region of the cloned TH2B gene contained S-phase-specific transcription elements which regulated expression of a reporter gene in an S-phase-specific manner. The S-phase-regulatory element was found to be located in two regions containing CCAAT elements between -153 and -110 base pairs (bp) and an octamer element (ATTTGCAT) between -109 and -84 bp. The two regions were required for a maximal stimulation of transcription of the cloned TH2B gene in S phase. On the other hand, only the octamer element was reported be important for the S-phase-specific transcription of human H2B gene. Since the synthesis of the TH2B histone is independent of DNA synthesis and specific for pachytene spermatocytes in vivo, the presence of the S-phase-specific transcription regulatory elements in the TH2B gene is surprising.During S phase in most eucaryotic cells, newly replicated DNA is assembled into chromatin by histone proteins (36). Therefore, there is a great demand for histones during S phase, when DNA replication occurs. Cells meet the demand for histone proteins by two different mechanisms: increased expression of histone genes and stabilization of histone mRNAs during the S phase (19,29,30,35,41,44). In this manner, cells achieve more than a 10-fold increase in histone mRNA during the S phase. The coupling of histone and DNA synthesis has been well established, but the mechanism of coupling is not clearly understood. Recently attention has been focused on the molecular mechanisms of the cell cycle-dependent expression of histone genes and the stabilization of mRNAs during S phase. The S-phase-specific histone gene transcription appears to be regulated by cisacting DNA elements present 5' of histone genes (3,23,31,40,42). Also, histone mRNAs appear to have a sequence element(s) for stabilization of the mRNAs during the S phase of the cell cycle (1,7,29,39,44). The 3' noncoding region of the histone mRNA has a highly conserved hairpin structure that appears to influence S-phase-specific stability of histone mRNAs (7, 39, 44).There are histone variants which are cell cycle-independent and cell type specific. One of the cell cycle-independent variants is the human H3.3, which has an intron and encodes polyadenylated mRNA (46). The best-known examples of cell type-specific histones are the avian erythrocyte-specific H5 (11, 45) and the mammalian testis-specific histones (4, 27). During spermatogenesis, testis-specific histones (TH1, TH2A, and TH2B) are synthesized in the mammalian testis in the absence of DNA replication, and these histones replace ...