The testis-specific histone gene H1t is expressed only in mammalian testis at the stage of pachytene spermatocytes. The tissue-specific regulation of the mouse H1t gene was examined in mouse testicular primary culture cells with gene constructs consisting of H1t promoter elements fused to the chloramphenicol acetyltransferase or the firefly luciferase reporter gene. Our experiments demonstrate that expression of the mouse H1t gene is enhanced by a conserved H1 histone gene-specific TG box 452 base pairs upstream of the transcription start site. The transcription of the H1t gene appears to be reduced by sequences between -1999 and -1506. No regulatory effect could be shown for the H1 box in the expression of the mouse H1t gene. Binding of nuclear protein extracted from mouse testis to these consensus elements was shown by electrophoretic mobility-shift assays with mouse testicular nuclear proteins and labeled oligonucleotides containing the upstream TG box sequences or the two testis-specific elements of the H1t gene.