We have produced a fine restriction map around the locus D4F104S1 (previously designated D4S810); a probe to this locus, p13E-11, identifies a polymorphic EcoRI fragment containing 3.2kb tandem repeats and detects DNA rearrangements associated with facioscapulohumeral muscular dystrophy (FSHD). We developed an STS (D4F106S1) which maps 2kb proximal to D4F104S1, and used this to isolate a 470kb YAC (y25C2E) from the ICI YAC library and a 930kb YAC (y956A11) from the CEPH megabase library. Both YACs contain the loci D4S139, D4F35S1 and D4F104S1. A cosmid library was produced from YAC y25C2E and two cosmid contigs constructed; a 115kb contig encompassing D4S139, and one of 135kb linking D4F35S1 and D4F104S1 and extending distal to the EcoRI fragment detected by p13E-11. A fine restriction map of both these contigs has been generated, allowing the orientation of the EcoRI fragment rearranged in FSHD to be determined. YAC y956A11 was used to confirm the integrity of y25C2E and the map of this region. 9B6A, a probe to the homeobox region of the tandem repeat D4Z4, identified a cross-hybridising sequence proximal to D4F104S1, however, p13E-11 does not detect this additional locus. CpG islands were identified between D4S139 and D4F35S1 and within each copy of the tandem repeat. The probe 9B6A detected each copy of the repeat motif, suggesting there is homeobox present in every copy of the 3.2kb repeat.