Deletions involving the chromosome 9p21 region have been reported as frequent events in non-small cell lung cancer (NSCLC). To investigate potential tumor-suppressor gene (TSG) loci within the 9p21 region, which also harbors the candidate TSG locus CDKN2a, we studied 32 cases of primary NSCLC for loss of heterozygosity (LOH). Tumor and paired normal lung cells were microdissected from lung tissue imprints and all samples screened using PCR-LOH analysis with 15 9p markers. In addition, 3 NSCLC cell lines and their matched normal lung and tumor DNA were similarly analyzed. LOH at the marker D9S259, which is proximal to the CDKN2a locus, was found most frequently (52%), while LOH at D9S942, the marker closest (5 kb) to the CDKN2a gene, was seen in only 17%. Homozygous loss of markers close to CDKN2a was, however, detected in 2 of the 3 cell lines and one accompanying tumor sample. We propose that a TSG in the region of deletion proximal to the CDKN2a gene within 9p21 may play a significant role in the pathogenesis and progression of NSCLC.