The phosphoglycerate kinase-2 (Pgk2) gene is regulated in a tissue-, cell type-, and developmental stage-specific manner during spermatogenesis and is required for normal sperm motility and fertility in mammals. Activation of Pgk2 transcription is regulated by testis-specific demethylation of DNA and binding of testis-specific transcription factors to enhancer and core promoter elements. Here, we show that chromatin remodeling including reconfiguration of nucleosomes and changes in histone modifications is also associated with transcriptional activation of the Pgk2 gene during spermatogenesis. Developmental studies indicate that the order of events involved in transcriptional activation of the Pgk2 gene includes demethylation of DNA in T₁- and T₂-prospermatogonia, binding of a factor to the CAAT box in type A and B spermatogonia, followed by recruitment of chromatin remodeling factors, displacement of a nucleosome from the Pgk2 promoter region, binding of factors to the Pgk2 core promoter and enhancer regions, and, finally, initiation of transcription in primary spermatocytes. Transgene studies show that Pgk2 core promoter elements are required to direct demethylation of DNA and reconfiguration of nucleosomes, whereas both enhancer and core promoter elements are required to direct changes in histone modifications and initiation of transcription. These results provide novel insight into the developmental order of molecular events required to activate tissue-specific transcription of the Pgk2 gene, the distinct elements in the 5'-regulatory region of the Pgk2 gene that regulate each of these events, and the relationship among these events in that each step in this process appears to be a necessary prerequisite for the subsequent step.