During spermatogenesis the X-chromosome is inactivated, but several glycolytic cycle enzymes have been found to have spermatogenic cell specific isoforms encoded by the X-chromosome counterpart genes located on the autosomes. Here, the promoters of the human glycerol kinase gene family were cloned: the somatic gene called GK-Xp, and the two testis-specific isoforms called GK-1 and GK-2, using Alu-GSP primers. The structure and function of the promoters were determined by sequencing and in situ expression studies using GFP as reporter. The transcription initiation sites were mapped by RACE and, these were found at -112 for GK-Xp, -46 for GK-1, and for GK-2 at -57 nucleotide positions, upstream of the translation start codon. The GK-Xp promoter specifically contains a TATA-like motif at -26, and a CCAAT motif at -149, which are not found in either the GK-1 or GK-2 promoters. Interestingly, GK-1 contains an Inr motif, a Sp1 binding site at -11, and a DPE motif at +27 positions, whereas GK-2 contains an Inr motif, a CRE at -11, a DPE at +29 and a RARE motif at +43 positions. In addition, they both contain MEP-2 and Ets motifs, in common with other testis-specific metabolic enzymes genes, such as PGK-2 and PDHA-2.