We have analyzed the occurrence of transcripts produced from the ubiquitously expressed, X-linked Pgk-1 gene and the testis-specific, autosomal Pgk-2 gene during spermatogenesis in the mouse. We found that tissue specificity, developmental specificity, and cell-type specificity of these mRNAs parallel that previously reported for the two protein isozymes of phosphoglycerate kinase (PGK) encoded by these two genes. This indicates that primary regulation of differential expression of the Pgk genes during spermatogenesis is exerted at the transcriptional level. We first detected Pgk-2 mRNA in preleptotene spermatocytes, indicating that transcription of Pgk-2 is initiated coincident with the onset of meiosis in male germ cells, and then continues to increase in later spermatocytes and postmeiotic round spermatids. This expression initiates prior to an initial decline in Pgk-1 transcript levels observed in pachytene spermatocytes, which apparently follows inactivation of the single X chromosome in spermatogenic cells. However, unlike cessation of Pgk-1 transcription from the inactivated X chromosome in female somatic cells, we show that inactivation of the Pgk-1 locus in spermatogenic cells is not followed by methylation of a key CpG dinucleotide in the promoter region. These results support the idea that specific expression of the Pgk-2 gene in meiotic and postmeiotic spermatogenic cells has evolved to compensate for reduced levels of Pgk-1 gene product caused by transient X-chromosome inactivation in these cells. They further suggest that reinitiation of transcription of the paternal Pgk-1 allele shortly after fertilization is facilitated by constitutive hypomethylation in the promoter region of this gene throughout spermatogenesis.