We used overlapping and nested homozygous deletions, contig building, genomic sequencing, and physical and transcript mapping to further define a approximately 630-kb lung cancer homozygous deletion region harboring one or more tumor suppressor genes (TSGs) on chromosome 3p21.3. This location was identified through somatic genetic mapping in tumors, cancer cell lines, and premalignant lesions of the lung and breast, including the discovery of several homozygous deletions. The combination of molecular manual methods and computational predictions permitted us to detect, isolate, characterize, and annotate a set of 25 genes that likely constitute the complete set of protein-coding genes residing in this approximately 630-kb sequence. A subset of 19 of these genes was found within the deleted overlap region of approximately 370-kb. This region was further subdivided by a nesting 200-kb breast cancer homozygous deletion into two gene sets: 8 genes lying in the proximal approximately 120-kb segment and 11 genes lying in the distal approximately 250-kb segment. These 19 genes were analyzed extensively by computational methods and were tested by manual methods for loss of expression and mutations in lung cancers to identify candidate TSGs from within this group. Four genes showed loss-of-expression or reduced mRNA levels in non-small cell lung cancer (CACNA2D2/alpha2delta-2, SEMA3B [formerly SEMA(V), BLU, and HYAL1] or small cell lung cancer (SEMA3B, BLU, and HYAL1) cell lines. We found six of the genes to have two or more amino acid sequence-altering mutations including BLU, NPRL2/Gene21, FUS1, HYAL1, FUS2, and SEMA3B. However, none of the 19 genes tested for mutation showed a frequent (>10%) mutation rate in lung cancer samples. This led us to exclude several of the genes in the region as classical tumor suppressors for sporadic lung cancer. On the other hand, the putative lung cancer TSG in this location may either be inactivated by tumor-acquired promoter hypermethylation or belong to the novel class of haploinsufficient genes that predispose to cancer in a hemizygous (+/-) state but do not show a second mutation in the remaining wild-type allele in the tumor. We discuss the data in the context of novel and classic cancer gene models as applied to lung carcinogenesis. Further functional testing of the critical genes by gene transfer and gene disruption strategies should permit the identification of the putative lung cancer TSG(s), LUCA, Analysis of the approximately 630-kb sequence also provides an opportunity to probe and understand the genomic structure, evolution, and functional organization of this relatively gene-rich region.