The testis-specific histone H1t gene is transcribed exclusively in primary spermatocytes during spermatogenesis. Studies with transgenic mice show that 141 base pairs (bp) of the H1t proximal promoter accompanied with 800 bp of downstream sequence are sufficient for tissue-specific transcription. Nuclear proteins from testis and pachytene spermatocytes produce footprints spanning the region covering the repressor element (RE) from 100 to 125 nucleotides upstream of the H1t transcriptional initiation site. Only testis nuclear proteins bind to the 5′-end of the element and produce a unique, low-mobility complex in electrophoretic mobility shift assays. This testis complex is distinct from the complex formed by a repressor protein derived from several cell lines that binds to the 3′-end of the element. The testis complex band is formed when using nuclear proteins from primary spermatocytes, where the H1t gene is transcribed, and band intensity drops 70%–80% when using nuclear proteins from early spermatids, where H1t gene transcription ceases. Protein-DNA cross-linking experiments using testis nuclear proteins produce electrophoretic bands of 59, 52, and 50 kDa on SDS/PAGE gels.