Fifty-nine NotI linking clones have been isolated from a flow-sorted chromosome 22 cosmid library and mapped using fluorescence in situ hybridization and/or a panel of somatic cell hybrids. Fourteen clones map to the short arm of chromosome 22, 31 to the long arm, and 9 to other chromosomes; 5 clones could not be unambiguously mapped. To identify potentially informative genetic markers, the chromosome 22 clones were screened for poly(CA) sequences; 24 positively hybridizing clones, 10 on the long arm and 14 on the short arm, were identified. These clones will be useful for constructing a long-range restriction map of chromosome 22 and may facilitate the cloning of chromosome 22 genes.