A region-specific library for human chromosome 2p23-p25 was constructed using microdissection and polymerase chain reaction (PCR)-mediated microcloning techniques. This library is large, comprising 300,000 recombinant microclones. The insert sizes range between 50-600 base pairs (bp) with a mean of 200 bp. About 50%-60% of the clones contain unique or very low copy number sequence inserts as determined by their weak or no hybridization to total human DNA. A subset of 48 microclones that did not hybridize to total human DNA after colony hybridization was analyzed, and 26 (54%) clones were shown to contain single-copy inserts and hybridize to human chromosome 2 DNAs, indicating that they are human chromosome 2 specific. The human genomic fragments identified by these clones after cleavage with HindIII have also been characterized. The single-copy microclones were used to analyze an interstitial deletion in the 2p23.3-p25.1 region--46,XY, del(2) (pter-->p25.1::p23.3-->qter)--previously reported in a patient with severe growth and mental retardation and multiple anomalies. Of the 26 microclones analyzed, 14 clones were mapped to the deletion region. The availability of the 2p23-p25 region-specific library and the probes derived from the library should be valuable for fine structure physical mapping analysis and the cloning of disease-related genes localized to the region. These studies also demonstrate the efficiency with which useful probes can be quickly generated for genome studies and for positional cloning.