Trp53-deficient mice spontaneously develop lymphomas, mainly of thymic origin, although the molecular mechanism remains largely unknown. As several interaction effects between p53 and iNOS have been reported, we hypothesized that iNOS activity in the thymus is causally linked to lymphomagenesis in Trp53-deficient mice. We therefore created mouse strains with different combinations of the Trp53 and iNOS genes. Western blot and histologic analyses showed that the iNOS protein was constitutively expressed in the thymus independently of Trp53 status and its expression was enhanced in Trp53+/– and Trp53–/– mice compared to Trp53+/+ mice. Homozygous disruption of iNOS decreased the incidence of thymic lymphomas by almost 40% (p = 0.087) and 90% (p < 0.05) in Trp53–/– and Trp53+/– mice, respectively, compared to the respective iNOS wild-type mice but significantly (p < 0.05) increased the development of nonthymic lymphomas in Trp53–/– and Trp53+/– mice. Although iNOS gene disruption did not affect the phenotype of thymic lymphomas, absence of the iNOS gene shifted the spectrum of nonthymic lymphoma from the B-cell to the T-cell lineage. RT-PCR analysis revealed enhanced expression of IL-10, which could have a promoting effect on lymphomagenesis, even without any stimulation, in the spleen of aging mice with the gene combinations Trp53–/–iNOS–/– and Trp53+/–iNOS–/– but not Trp53–/–iNOS+/+ or Trp53+/–iNOS+/+. These results suggest that iNOS could increase the development of thymic lymphomas in Trp53-deficient mice. While iNOS may have protective effects against nonthymic lymphomagenesis, the regulation of cytokine production by iNOS may be involved in the underlying mechanism of antilymphomagenesis effects in the peripheral lymphoid organ. © 2004 Wiley-Liss, Inc.