We tested 519 chromosome 3-specific cosmids for the presence of rare restriction-endonuclease sites in a search for cosmids containing HTF islands. We have identified 49 cosmids (9% of those tested) that contain multiple rare restriction-endonuclease sites. The cosmids were digested with several common cutting restriction endonucleases to liberate small fragments which were tested as unique-sequence chromosome 3-specific hybridization probes and for evolutionary sequence conservation. Unique-sequence hybridization probes isolated from the cosmids were hybridized to a somatic cell hybrid deletion mapping panel to subchromosomally localize the cosmids. Fragments from many of these cosmids demonstrated conservation of sequence through evolution, and these fragments hybridize to distinct transcripts. These cosmids should therefore prove a useful resource for the identification of many chromosome 3-specific genes, in addition to having potential use as linking clones for pulsed-field gel mapping studies.