Homozygous deletions are instrumental in the detection and cloning of tumor suppressor genes. We report the isolation and characterization of 39 new single-copy probes saturating a submicroscopic homozygous deletion detected in the DNA of the small cell lung cancer (SCLC) cell line U2020. The probes were selected from a large collection, covering the entire length of chromosome 3 with an estimated average spacing of 100-150 kb. Based on the number of probes in the deletion and the probe density, the size of the U2020 submicroscopic deletion was estimated to be in the range of 4-7 megabases. Among the deleted loci, 17 showed conservation across species, probably representing potential coding gene sequences. By genetic and physical mapping of a large randomly chosen fraction of the deleted probes, we defined the location of the U2020 deletion within chromosome band 3p12. Our cloning strategy is based on narrowing the region of interest by eliminating probes that retain heterozygosity in SCLC samples, thus selecting for probes in the region of common loss.