AbstractThe testis‐specific linker histone H1t gene is transcribed exclusively in pachytene primary spermatocytes. Tissue specific expression of the gene is mediated in part by transcriptional factors that bind elements located within the proximal and distal promoter. A 40 bp promoter element, designated H1t/TE, that is located within the proximal promoter between the CCAAT‐box and AC‐box, is known to be essential for H1t gene transcription in transgenic animals. In the present study, we show by SDS–PAGE analysis of UV crosslinked protein and DNA and by electrophoretic mobility shift assays (EMSA) of testis nuclear proteins separated on a non‐denaturing glycerol gradient that the TE1 sub‐element is bound by a protein complex. Mutation of TE1 leads to a drop in H1t promoter activity in germinal GC‐2spd cells as well as in nongerminal Leydig, NIH3T3, and C127I cell lines. Although TE1 and TE2 sub‐elements have similar sequences, mutation of the TE2 sub‐element causes an increase in promoter activity in C127I and Leydig cells. The rat TE1 but not TE2 contains a CpG dinucleotide and this cytosine is methylated in liver but not in primary spermatocytes. Methylation of the cytosine at this site almost eliminates nuclear protein binding. Thus, there are significant functional differences in the TE2 and TE1 sub‐elements of the H1t promoter with TE1 serving as a transcriptional activator binding site and TE2 serving as a repressor binding site in some cell lines. J. Cell. Biochem. 88: 1177–1187, 2003. © 2003 Wiley‐Liss, Inc.