Facioscapulohumeral muscular dystrophy (FSHD) is caused by deletions of 3.3-kb tandemly repeated units contained within a large polymorphic EcoRI fragment close to the telomere of chromosome 4q. Since the rearrangements were assumed to interfere with the structure or function of the putative FSHD gene, the gene search was focused on cosmids containing these repeat units and, in addition, cosmids spanning 75 kb of upstream sequences. cDNA selection hybridization was applied to four overlapping cosmid clones, yielding a total of 150 putative cDNA clones. These clones showed a random distribution across the cosmid contig, except for three regions which contained a much larger number of clones. Nine cDNA clones hybridized to a 2.2-kb EcoRI fragment, located 22 kb centromeric to the 3.3-kb repeated units. This 2.2-kb fragment showed evolutionary conservation, and analysis of the sequence by "GRAIL" predicted the presence of several exons. Transcripts homologous to this fragment could be identified but none of them originated from the 4q35 locus. Strikingly, most clones revealed 4-10 homologous loci, and no single copy clones could be isolated. These findings are in line with earlier observations by fluorescent in situ hybridization (FISH) showing hybridization of individual cosmid clones to multiple chromosomes. The presence of homologous regions on other chromosomes seriously complicates the cloning of the FSHD gene.