A somatic cell hybrid mapping panel that defines seven regions of the long arm and one region of the short arm of human chromosome 6 has been developed. Utilizing this panel, 17 NotI boundary clones from a NotI linking library were regionally assigned to the long arm of chromosome 6. The majority of these clones (11) were found to localize within band regions 6q24-q27. The nonuniform distribution of NotI sites may indicate a cluster of HTF islands and likely represents a coincidence of coding sequences in this region of chromosome 6. Cross-hybridization of these linking clones to DNA from other species (zoo blots) provides further evidence for transcribed sequences in 7 of the NotI clones. These NotI clones were also used to identify corresponding NotI fragments using pulsed-field gel electrophoresis, facilitating further physical mapping of this region. Finally, regional assignment of five polymorphic probes to the long arm of chromosome 6 is also presented. These hybrids and probes should facilitate the construction of a physical and genetic linkage map to assist in the identification of disease loci along chromosome 6.