Phosphoglycerate kinase (PGK), a major enzyme in glycolysis, is encoded by the X chromosome in mammals. We have initiated molecular analysis of the PGK structural gene by isolating a full-length cDNA clone from a human fetal liver cDNA library. Synthetic oligonucleotide mixtures encoding two different portions of PGK were used as direct in situ hybridization probes for the cDNA library. Several classes of clones were obtained based on their hybridization at different stringencies to one or both of the PGK oligonucleotide mixtures. One clone, designated pHPGK-7e, which hybridized at high stringency to each of the synthetic probes, encoded the complete PGK protein sequence as well as 82 base pairs of 5' and 437 base pairs of 3' untranslated regions. Southern blot analysis of human genomic DNAs revealed a complex pattern of hybridizing fragments, two of which were non-X in origin. These results suggest that the human genome contains a small family of dispersed PGK or PGK-like genes.