We report the isolation and characterization of a polymorphic pentanucleotide repeat (TAAAA)n, which was mapped to human chromosomes X and Y (loci DXYS156X and DXYS156Y) by PCR amplification of DNA from a monochromosomal somatic cell hybrid panel (NIGMS panel 2). The (TAAAA)n repeat of loci DXYS156 occurs within a human LINE element at a position where the consensus sequence contains a single TAAAA motif. In 72 unrelated CEPH individuals seven alleles were detected which ranged in size from 125 to 165 bp in 5 bp intervals. The two largest alleles (160 and 165 bp) were observed only in males, which suggests that they were amplified from the Y chromosome DXYS156Y locus. The other 5 alleles were present in two copies in females and in a single copy in males, which suggests that they were amplified from the X chromosome DXYS156X locus. Locus DXYS156X was polymorphic in CEPH families with an observed heterozygosity in females of 46% (27 of 59). Linkage analysis with DNA markers on the X chromosome revealed significant lod scores for a location of DXYS156X close to markers DXS1002 (theta = 0.000; zeta = 8.43), DXYS1X (theta = 0.015; zeta = 17.3), DXS3, and PGK1 in the region of chromosome Xq13. The sequence of DXYS156Y derived from the 165 bp allele has been deposited in Genbank with accession number X71600.