The identification of a tumor suppressor gene in non-small cell lung cancer (NSCLC) is one of the most important issues to elucidate the molecular mechanisms of this type of refractory cancer and to establish a novel strategy against it. Since NSCLC, like most other human cancers, develops as a sporadic disease, linkage analysis is not available for gene cloning. This review describes the functional cloning approaches to a tumor suppressor gene in sporadic cancers. Suppression of the malignant phenotype of cancer cells by fusion with a normal fibroblast was the first demonstration of the recessive phenotype of cancer cells in 1969. Evidence of tumor suppressor genes on the specific chromosomes was later provided by functional complementation of the cancer phenotype through microcell-mediated chromosome transfer. Further introduction of more restricted DNA fragments by YAC transfer provides a potent tool to localize the gene to a small segment, appropriate for the subsequent gene cloning. TSLC1, a novel tumor suppressor gene in NSCLC, was identified on chromosome 11q23. 2 through a series of functional complementation of A549 cells in tumorigenicity. Two-hit inactivation of the TSLC1 by promoter methylation and gene deletion was observed in 40% of primary NSCLC tumors. The strong tumor suppressor activity of TSLC1, and its possible involvement in cell adhesion, suggest that the functional cloning approach could cast a new light on a group of genes that have not yet been characterized, but are important for general human carcinogenesis as well as tumor suppression.