Serotonin (5HT) was shown to induce in vitro the production of ROS in the presence of neuronal nitric oxide synthase (nNOS) in addition to the basal NO(+) formation. With the aim of understanding this mechanism, this study investigated the potential binding of 5HT to nNOS. By using [(3)H]5HT, it is reported here that 5HT binds to nNOS, but only when the enzyme is active and in a superoxide-dependent manner. This binding is prevented by DPI but not by L-NAME. The formation of 5HT-nNOS complex was shown to be very well correlated with the production of ROS by 5HT in the presence of nNOS. A mechanism involving nNOS only in its initial step is proposed to explain both the formation of 5HT-nNOS complex and the production of ROS observed in the presence of nNOS and 5HT.