High risks of lung cancer occur after successful treatment of Hodgkin's disease. In addition to tobacco smoking, other risk factors include radiotherapy, chemotherapy, and immunosuppression, although the relative contributions of each are unknown. We conducted p53 mutational spectrum analysis in second lung cancers after radiation therapy for Hodgkin's disease in the Netherlands and in Ontario, Canada. Lung cancer tissues from 11 patients were analyzed by p53 immunohistochemistry and DNA sequence analysis. All were male cigarette smokers, all received radiation therapy, and six also received chemotherapy. The lung cancers occurred 9.8 years (mean) after treatment. Radiation doses to lung lobes that developed the tumors averaged 5.7 Gy (range, 3.7-11.7 Gy). Sequence analysis showed four missense and two silent p53 point mutations in five patients. There were four G:C-->A:T transitions; three of four mutated deoxyguanines occurred on the coding strand, and one was a CpG site. There were two transversions: one G:C-->C:G and one A:T-->C:G. Despite moderate or heavy smoking histories in all patients, the mutational spectrum appears to differ from usual smoking-related lung cancers in which G:C-->T:A transversions predominate. The absence of G:C-->T:A mutations and the prominence of G:C-->A:T transitions, which are characteristic of radiation and oxidative damage, suggest that radiotherapy might have caused some of the p53 mutations. These data illustrate the potential of mutation analysis to determine causes of human cancer. If confirmed in a larger series, these results imply that some radiation-induced cancers can be distinguished from those caused by other factors.