The frequent occurrence of 21q deletions in human non-small cell lung carcinoma (NSCLC) indicates the presence of a tumor suppressor gene on this chromosome arm. Since the ANA (Abundant in Neuroepithelium Area) gene, a member of an antiproliferative gene family, was mapped to 21q11.2-q21.1, we searched for genetic alterations of the ANA gene in human lung cancers. The gene was homozygously deleted in a human NSCLC cell line, Ma17. The gene was mapped in the 0.33 Mb Not1 fragment at 21q21.1 of the Not1 restriction map for 21q. Loss of heterozygosity (LOH) at this locus was detected in 24/47 (51.1%) of NSCLC, and the frequency of LOH in brain metastases was significantly higher than that in stage I-II primary tumors (P = 0.018). These results suggested that the homozygously deleted region harbors a novel tumor suppressor gene involved in NSCLC progression. Since mutation of the ANA gene was not detected in other lung cancer cell lines and fresh lung tumors with LOH at this locus, it is unlikely that the ANA gene is a target gene inactivated by two mutational events in this chromosomal region. Physical mapping of the homozygously deleted region showed that the deletion had occurred interstitially at 21q11.1-q21.1 and the size of the deletion was estimated as being more than 3 Mb. Our mapping results will facilitate further efforts to identify a tumor suppressor gene on 21q.