The pseudoautosomal (PA) region of the mammalian genome is the region of the X and Y chromosomes that shares extensive DNA sequence homology and is of special interest because it may play an essential role during male meiosis. We have identified three telomere-related restriction fragments from the PA region of the mouse genome, using an oligonucleotide probe composed of the mammalian telomere consensus sequence TTAGGG. PA assignment of two C57BL/6J-derived fragments was initially suggested by analysis of DNAs from progeny sired by C57BL/6J males carrying the rearranged Y chromosome, Y*: the hybridization intensity of both fragments was concordant with the sex-chromosome complement of the offspring. Further analysis indicated that both fragments were present in female and male F1 mice regardless of the sex oftheir C57BL/6J parent-a criterion for autosomal or PA linkage. Both fragments were closely linked to each other and located on the X chromosome distal to amelogenin (Amg)-in agreement with X or PA linkage. Confirmation of the PA derivation of these fragments was accomplished by following their segregation in a cross involving XY* males mated to DBA/2J females. A similar experiment identified a third PA-derived restriction fragment of LT/SvEi origin. Identification of PA-derived telomere-related restriction fragments will enable further genetic analysis ofthis region of the mouse genome.In 1967, Ohno (1) suggested that the mammalian X and Y chromosomes are derived from a pair of homologous chromosomes, an extension of an hypothesis proposed by Muller (2) for the origin of the sex chromosomes of Drosophila. If this is true, then numerous rearrangements, additions, and deletions must have occurred in the ancestral chromosome that evolved into the mammalian Y chromosome, as the Y chromosome now differs significantly from its putative evolutionary homologue, the X chromosome. However, one area of extensive homology still exists between the mammalian X and Y chromosomes. This shared segment is commonly referred to as the pseudoautosomal (PA) region because PA-derived loci are inherited in a manner characteristic of autosomal loci as a result of obligate crossing over between the X and Y chromosomes during male meiosis (3). In humans the PA region is located at the distal end of the short arms of the X and Y chromosomes, whereas in mice this region is located at the distal end ofthe long arms ofthe X and Y chromosomes (3,4).Although a number of DNA sequences and loci have been mapped to the PA region of the human genome (e.g., ref. 5), to our knowledge only three loci have been mapped to the PA region of the laboratory mouse genome: steroid sulfatase