The aim of this prospective study was to determine whether use of a combination of biomarkers, p53 and nuclear DNA content, led to improved prognosis and clinicopathologic correlation in human non-small cell lung cancer. Nineteen patients undergoing curative resection of primary non-small cell lung cancer were evaluated. Resected tumors were studied by polymerase chain reaction/single strand conformation polymorphism analysis (p53 gene mutations), flow cytometry (nuclear DNA content and cell cycle analysis), and immunohistochemically (p53 oncoprotein). Histologically normal lung was used as an internal control for each patient. Minimum postoperative follow-up was 4 years. p53 gene mutations (5/19 tumors; 26%), tumor ploidy (5/19 diploid), patterns of immunoreactivity, or combination of biomarkers did not appear to correlate with clinicopathologic findings or clinical outcome. Two of three patients with associated second primary malignancies, had squamous cell diploid tumors with p53 gene mutations. We conclude that p53 gene mutations and tumor ploidy may represent different biologic markers for human non-small cell lung cancer. Although trends in improved predictive accuracy were not seen when both markers were incorporated into the tumor analysis, flow cytometry and molecular analysis of the p53 gene may identify patients at increased risk of the development of a second primary malignancy.