Diesel exhaust particles (DEP) induce pulmonary tumors, asthma-like symptoms, and the like in experimental animals. The involvement of reactive oxygen species (ROS) is suggested in the injuries induced by DEP, though the generation of ROS has not been proven. The present study provided the first direct evidence of *OH generation in the lungs of living mice after intratracheal instillation of DEP, using noninvasive L-band ESR spectroscopy and a membrane-impermeable nitroxyl probe. *OH generation is confirmed with the enhancement of in vivo ESR signal decay rate of the probe. The decay rate at mid-thorax was significantly enhanced in DEP-treated mice compared to that in vehicle-treated mice. The enhancement was completely suppressed by the administration of either *OH scavengers, catalase, or desferrioxamine, while the administration of SOD further increased the rate. The administration of Fenton's reagents into the lung also enhanced the decay rate of the probe at mid-thorax of mice. These results clearly provided evidence that the intratracheal exposure to DEP in mice produced *OH in the lung through an iron-catalyzed reaction of superoxide/H(2)O(2). This first direct evidence of *OH generation in DEP-treated mice lung may be utilized to determine treatments for DEP-induced lung injury.