During spermatogenesis, approximately 85% of histones are replaced by protamines. The remaining histones have been proposed to carry essential marks for the establishment of epigenetic information in the offspring. The aim of the present study was to analyse the expression pattern of histone H3 acetylated at lysine 9 (H3K9ac) during normal and impaired spermatogenesis and the binding pattern of H3K9ac to selected genes within ejaculates. Testicular biopsies, as well as semen samples, were used for immunohistochemistry. Chromatin immunoprecipitation was performed with ejaculated sperm chromatin. HeLa cells and prostate tissue served as controls. Binding of selected genes was evaluated by semiquantitative and real-time polymerase chain reaction. Immunohistochemistry of H3K9ac demonstrated positive signals in spermatogonia, spermatocytes, elongating spermatids and ejaculated spermatozoa of fertile and infertile men. H3K9ac was associated with gene promoters (CRAT, G6PD, MCF2L), exons (SOX2, GAPDH, STK11IP, FLNA, PLXNA3, SH3GLB2, CTSD) and intergenic regions (TH) in fertile men and revealed shifts of the distribution pattern in ejaculated spermatozoa of infertile men. In conclusion, H3K9ac is present in male germ cells and may play a role during the development of human spermatozoa. In addition, H3K9ac is associated with specific regions of the sperm genome defining an epigenetic code that may influence gene expression directly after fertilisation.