Human sperm, unlike the sperm of other mammals, contain replacement histones with unknown biological functions. Here, we report the identification of the novel human gene coding for a testis/sperm-specific histone H2B (hTSH2B). This variant histone is 85% homologous to somatic H2B and has over 93% homology with the testis H2B of rodents. Using genomic PCR, two genetic alleles of hTSH2B were found in the human population. The hTSH2B gene is transcribed exclusively in testis, and the corresponding protein is also present in mature sperm. We expressed recombinant hTSH2B and identified this protein with a particular H2B subtype expressed in vivo. The subnuclear distribution of H2B variants in sperm was determined using biochemical fractionation and immunoblotting. The H2B variant associated with telomere-binding activity () was solubilized by Triton X-100 or micrococcal nuclease extraction, whereas hTSH2B was relatively tightly bound in nuclei. Immunofluorescence showed that hTSH2B was concentrated in spots located at the basal nuclear area of a subpopulation (20% of cells) of mature sperm. This fact may be of particular importance, because the hTSH2B "positive" and "negative" sperm cells may undergo significantly different decondensation processes following fertilization.