Eight single-copy DNA probes specific for human chromosome 3 were isolated by screening a human chromosome 3-derived genomic library. Southern blot analyses of DNAs isolated from a panel of somatic cell hybrids allowed us to regionally assign all probes to subregions on chromosome 3. Three clones were localized to the short arm of chromosome 3 (3p21→pter), two to the long arm (3q21→qter), and three to the 3q21→3p21 sub-region. Six of these DNA sequences map to regions overlapping a segment of chromosome 3 (3p14→p23) frequently deleted in small cell lung cancer cells. Restriction fragment length polymorphism analyses indicate that at least three of the eight single-copy probes studied show Msp1 or Bg/II polymorphisms. This library is a useful source of chromosome 3-specific