Although nitric oxide (NO) plays an important role in the pathophysiological process of cerebral ischemia or severe traumatic brain injury, its contribution to the pathogenesis of moderate diffuse axonal injury (mDAI) remains to be clarified. The alterations in nitric oxide synthase (NOS) activity and the histopathological response after mDAI was investigated. Forty anesthetized Sprague-Dawley adult rats were injured with a Marmarou's weight-drop device through a Plexiglas guide tube. These rats were divided into 8 groups (control, 1 hr, 2 hr, 3 hr, 6 hr, 12 hr, 24 hr, 48 hr after trauma). The temporal pattern of apoptosis in the adult rat brain after mDAI was characterized using TUNEL histochemistry. In addition, the cDNA for NOS activity was amplified using RT-PCR. The PCR products were electrophoresed on a 2% agarose gel. eNOS activity was not detected, but nNOS activity was expressed after 3 hr and continuously 48 hr after impact, which was approximately double that of the control group at 12 and 24 hr. Subsequently, there was a decrease in activity after 48 hr. The iNOS activity increased dramatically after 12 hr and was constant for a further 12 hr followed by a dramatic decrease below the level of the control group. Significant apoptotic changes occurred 12 and 24 hr. after insult. nNOS and iNOS activity were affected after moderate diffuse axonal injury in a time-dependent manner and there was a close relation between the apoptotic changes and NOS activity. Although the nNOS activity was expressed early, its activity was not stronger than iNOS, which was expressed later.