The histone H4t gene is actively transcribed in rat testis germinal cells and in several non-testis cell types including cells from liver. This histone H4 gene is expressed most actively in testis premeiotic pachytene spermatocytes, and its expression is down-regulated in postmeiotic early spermatids. The histone H4 gene promoter is functional as demonstrated by finding significant levels of chloramphenicol acetyltransferase (CAT) mRNA in mammalian cells transiently transfected with a histone H4-promoted CAT expression vector compared to cells transfected with an expression vector lacking of the promoter. Examination of the proximal promoter of the histone H4 gene by electrophoretic mobility shift assays indicates that specific protein-DNA interactions occur when pachytene spermatocyte nuclear proteins are mixed with promoter fragments containing regions designated site I and site II, consensus sequence elements essential for regulating transcription of the histone H4 gene. These specific protein-DNA interactions are eliminated by competition with identical DNA fragments, but are not eliminated by competition with nonhomologous DNA fragments. Significant differences are found in mobility shift patterns upon comparison of nuclear proteins from germinal cell populations enriched in pachytene spermatocytes where the gene is actively expressed and early spermatids where the gene is not expressed.