The status of the P16 gene was investigated by Southern blot, polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP), and DNA sequencing analyses in 30 primary resected non-small cell lung carcinomas (NSCLCs) with metastatic involvement of thoracic lymph nodes and 33 NSCLCs without node metastases. Direct sequencing of tumour DNA samples scored positive by PCR-SSCP showed five somatic mutations of the P16 gene: four nonsense and one frameshift. The Southern blot analysis revealed the presence of a homozygous deletion of the P16 locus in one tumour. All of the six NSCLCs with somatic aberrations of the P16 gene belonged to the series of tumours with metastatic diffusion to thoracic lymph nodes. In each of these six cases, the genetic aberration was seen in both the primary tumour and the node metastasis. No P16 alteration was found in tumours without metastatic lymph nodes. This difference was statistically significant (P = 0.02). No correlation was present between P16 alterations and other clinicopathological parameters including age of patients, tumour size, histological type, and grade. In three tumours with genetic aberration of P16, there was a concomitant alteration of the p53 gene. Our results indicate that the P16 gene is infrequently mutated (10 per cent of the cases examined) in primary resected NSCLC. However, since P16 mutations were found only in metastatic tumours, they may be important events in late phases of tumour progression and could represent useful markers of tumour aggressiveness in NSCLC.