The testis-specific histone H1t is synthesized during spermatogenesis exclusively in late pachytene primary spermatocytes. Transcription of the H1t gene is repressed in every tissue except testis. Within the testis, transcription is repressed during development before the spermatocyte stage and in later stages of germinal cell maturation. Mechanisms involved in transcriptional repression of the H1t gene are unknown. To assess the contribution of upstream H1t promoter sequence to transcriptional silencing in nonexpressing cells, H1t-promoted reporter vectors were constructed using pGL3 Basic. Transient expression assays with these reporter vectors driven by H1t promoter deletions allowed us to identify a region from 948 to 780 bp upstream from the H1t transcriptional initiation site that functions as a silencer. Examination of nuclear protein binding to this DNA regulatory region by electrophoretic mobility shift assays using extracts from C127I cells, rat testis, and pachytene spermatocytes revealed a low mobility band produced only by nuclear proteins derived from nonexpressing cells that may contain proteins that repress H1t gene transcription.