6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) catalyzes the synthesis and degradation of fructose-2,6-bisphosphate, a potent regulator of glycolysis. Previous studies assigned the gene for human liver PFK-2/FBPase-2 (HGMW-approved symbol PFKFB1) to the X chromosome; however, precise localization remained ambiguous, with the gene variously placed between Xcen-q13, Xq27-q28, and Xp11.22-p11.21. We have localized the gene within a YAC contig clustered around ALAS2 (human erythroid delta-aminolevulinate synthase) in Xp11.21 and have identified eight YACs positive for the gene. Four of these overlapping YACs were mapped using rare-cutter restriction enzymes to provide in-depth characterization of an 820-kb region encompassing the PFK-2/ FBPase-2 and ALAS2 genes. PFK-2/FBPase-2 was found to lie close (within approx. 250 kb) and telomeric to ALAS2. Three putative CpG islands were also detected in the region.