We analyzed the pattern of allelic loss on chromosome 9 in 40 primary human non-small cell lung cancers including 16 squamous cell, 18 adeno-, and 6 large cell carcinomas. Using 24 polymorphic microsatellite markers spanning chromosome 9, we found that 27 of 40 (67.5%) of these neoplasms displayed loss of heterozygosity (LOH) on chromosome 9. Most tumors showed LOH for all informative markers on both chromosomal arms, whereas five tumors demonstrated partial LOH on chromosome 9. In four of these tumors, allelic loss was limited to the 9p arm, whereas in the remaining specimen, LOH extended from 9p21-22 to terminal 9q. These five tumors delineate a minimal area of loss at 9p21-22, which includes a previously defined tumor suppressor gene locus. We have identified a distinct region of loss on chromosome 9p commonly involved in non-small cell lung cancer tumorigenesis.