We have established a protocol for producing libraries of specific mouse chromosomes. The mouse DNA-containing clones from a genomic library of a hamster-mouse somatic cell hybrid containing only one mouse chromosome are identified by screening with radiolabeled mouse repetitive sequences after specifically blocking hamster repetitive sequences; 95% of the mouse DNA-containing clones are identified. We have applied this protocol in producing a library of mouse chromosome 16, consisting of 14,200 clones or two "chromosome equivalents." Each clone occupies an individual well in a microtiter tray, allowing the entire library to be repeatedly and reproducibly plated and analyzed by hybridization. Further, we have established a protocol for making cDNA probes specifically depleted of highly repetitive sequences for probing libraries of genomic clones. By screening the chromosome 16 library with cDNA probes from mouse liver and brain, we demonstrate the feasibility of identifying expressed sequences and characterizing their patterns of expression. Such chromosome-specific libraries can facilitate the isolation of defined genetic loci as well as form the basis for the production of integrated transcriptional, genetic, and physical maps of entire chromosomes.