The human hydroxyindole-O-methyltransferase (HIOMT) gene has been mapped to the X chromosome using cDNA probes from the 3' and 5' regions of a human cDNA clone. Southern blot analysis of a panel of human-rodent somatic cell hybrid DNAs revealed that the gene was localized to the short arm of chromosome X, and most probably the pseudoautosomal region of the human X (Xp22.3) and Y (Yp11.3) chromosomes. Several multiallelic restriction fragment length polymorphisms were detected at this locus allowing further localization of the gene by two-point and multipoint linkage analysis in the 40 CEPH families. These results confirmed the pseudoautosomal localization of the HIOMT gene and allowed ordering of the gene in close proximity to DXYS17 at a position about 600-800 kb from the pseudoautosomal boundary and about 1800 to 2000 kb from the telomere. It will be possible to readily identify or exclude the involvement of this gene in genetic diseases by linkage analysis of the disease locus with the high frequency multiallelic polymorphisms at this locus.