The present study investigates the role of nitric oxide (NO) in the deletion of TCR-stimulated double-positive (DP) thymocytes. Fetal thymi expressed mRNA for an inducible type of NO synthase (iNOS). The levels of iNOS mRNA became maximal around gestation day 18 with a decline after birth. Administration of anti-CD3 mAb to fetal thymus organ culture (FTOC) or young mice resulted in enhanced expression of mRNAs for iNOS as well as IFN-gamma. Immunohistochemical analyses revealed that iNOS was produced in the corticomedullary junction and medulla. The effects of iNOS-induced NO on anti-CD3-unstimulated or anti-CD3-stimulated thymocytes were examined by culturing them in the presence or absence of a NO-generating compound. Stimulation of DP thymocytes with anti-CD3 alone induced the generation of CD4(low)CD8(low) thymocytes. The subsequent exposure of these anti-CD3-stimulated thymocytes to NO promoted down-regulation of CD4 and CD8 expression. The recovery of viable DP cells was considerably reduced compared with stimulation with anti-CD3 or NO alone. Even in a viable DP population, high incidences of DNA strand breaks were detected in the CD4(low)CD8(low) compartment. In contrast to DP cells, the recovery of viable single-positive cells was not decreased but rather slightly enhanced by treatment with anti-CD3 and/or NO. The recovery of anti-CD3-stimulated thymocytes were also reduced when cultured on the thymic stromal monolayer with the capacity to produce NO upon IFN-gamma stimulation. These results indicate that NO, which is generated in association with TCR stimulation in the thymus, functions to induce deletion of DP thymocytes, especially when their TCR is stimulated.