Genomic clones of human liver phosphofructokinase (PFK) were isolated by screening a gene bank enriched for chromosome 21 sequences with two synthetic oligonucleotide probes designed from peptide sequences of purified human liver PFK. A 3.3 Kb fragment derived from the genomic clones was sub-cloned and designated to pG-PFKL 3.3. It hybridized with a 3.5 Kb mRNA on Northern blots and was able to enrich selectively for liver PFK mRNA by hybrid-selection. These results demonstrated that the isolated clones contain sequences homologous to human PFKL mRNA. When hybridized to genomic DNA blots pG-PFKL 3.3 reacted with the same 3.3 Kb BamHI fragment in both human DNA and DNA of the mouse/human hybrid line WA17 which contains human chromosome 21 as the only human chromosome. These data confirm the assignment of the PFKL gene to chromosome 21.