Ikaros is a potent tumor suppressor gene for both human and murine leukemogenesis. Ikaros alteration in X-ray-induced thymic lymphoma (TL) was characterized by aberrant splicing, null expression and point mutation, which were accompanied by loss of heterozygosity (LOH) at Ikaros loci. N-ethyl-N-nitrosourea (ENU) -induced TL harbored only point mutation without LOH. We showed that combined treatment of X-rays (0.8-1.0 Gy x 4 times) and ENU (100, 200ppm) synergistically increased TL incidence. The aim of this study was to elucidate the underlying mechanism of the lymphomagenesis induced by combined treatment. B6C3F1 mice (4-week-old female) were irradiated with X-rays (0.2 - 1.0 Gy a week x 4 times) and then treated with ENU (50 - 200 ppm in drinking water) from 8-week-old. The analyses of LOH, expression and mutation of Ikaros were performed. The frequency of aberrant splicing, null expression and point mutation of Ikaros in X-ray-induced TL was 10, 12 and 26%, respectively. ENU-induced TL showed only point mutation in 26% of TL. LOH frequencies at Ikaros loci were 73% and 0% for X-ray and ENU-induced TL, respectively.TL after combined treatment rarely exhibited aberrant splicing and null expression. Surprisingly, point mutation was increased up to 40%, most of which did not accompany LOH, indicating similarity to those in ENU-induced TL. We conclude that X-rays play a role as a promoter for ENU-induced lymphomagenesis in combined exposure.