Homozygous deletions of the tumor suppressor gene p16 INK4A and deficiency of methylthioadenosine phosphorylase (MTAP), both located on chromosome 9p21, have been independently reported in non-small cell lung cancer (NSCLC). To determine the frequency of co-deletion of these two genes, we investigated 50 samples of primary NSCLC using a quantitative PCR-ELISA. All specimens were fixed in formalin, paraffin embedded and stored until assayed. Histologic subtypes included 25 adenocarcinomas (50%), 21 squamous cell carcinomas (42%) and four large cell carcinomas (8%). Homozygous deletions of MTAP exon 8 could be detected in 19 of 50 NSCLC samples (38%). Adenocarcinoma (11 of 25, 44%) showed a higher deletion frequency than squamous cell carcinoma (six of 21, 29%). In contrast, homozygous p16 INK4A deletions were detected in only nine of 50 (18%) samples using specific primers for p16 INK4A exon 1α. No difference between the histological subtypes and p16 INK4A deletion frequency was observed. We further investigated the ten samples with MTAP deletions but intact p16 INK4A exon 1α with primers specific for p16 INK4A exon 3, the exon nearest to MTAP exon 8. Interestingly, none of the ten samples had deletion of the p16 INK4A exon 3 coding region. Fine mapping analysis performed in ten samples showed a frequent breakpoint between MTAP exon 4 and exon 5. In addition, p16 protein expression could not be detected in five out of six samples with intact p16 but deleted MTAP locus. These data show a high frequency of homozygous MTAP deletions in NSCLC which is associated with detectable co-deletion of p16 INK4A in only half of the cases. This result suggests the existence either of another tumor suppressor gene telomeric of p16 INK4A or of deletions involving 3′-untranslated (3′-UTR) regulatory regions of p16 INK4A that can interfere with its expression or function.