K-ras mutation appears in about 60% of patients with non-small-cell lung cancer (NSCLC). This frequency and its presence in normal appearing tissues point to the potential of ras oncogene mutation to serve as a good biomarker. Using enriched PCR (EPCR), which enables the detection of one mutant allele in the presence of 10,000 normal alleles, we have determined the frequency of mutant ras alleles in the sputum samples of patients with or without lung cancer. Samples were collected from 17 patients with NSCLC and from 40 controls who suffered from non-oncological lung diseases, including bronchitis, asthma, and pneumonia. Of the 37 samples obtained from patients with lung cancer, 18 were found to harbor ras oncogene mutations (48%). Of the 40 cases that were free of lung cancer, five were found to harbor this mutation (12.5%). The difference between the two frequencies was found to be significant (P < 0.01). These findings indicate that (a) K-ras oncogene mutation can be identified in routinely obtained sputum samples of patients who may be at risk of developing lung cancer and (b) the higher frequency of these mutations in samples of patients with lung cancer points to the potential use of the ras mutation as a biomarker for either exogenous or endogenous exposure to carcinogens. Thus, the ability to examine sputum provides a powerful and convenient source of sampling and may be adapted for future large-scale screening.