Dopaminergic neurotransmission is involved in the regulation of sleep. In particular, the nigrostriatal pathway is an important center of sleep regulation. We hypothesized that dopaminergic neurons located in substantia nigra pars compacta (SNpc) could be activated by gentle handling, a method to obtain sleep deprivation (SD). Adult male C57/BL6J mice (N = 5/group) were distributed into non-SD (NSD) or SD groups. SD animals were subjected to SD once for 1 or 3 h by gentle handling. Two experiments were performed. The first determined the activation of SNpc neurons after SD, and the second examined the same parameters after pharmacologically induced dopaminergic depletion using intraperitoneal reserpine (2 mg/kg). After 1 or 3 h, SD and NSD mice were subjected to motor evaluation using the open field test. Immediately after the behavioral test, the mice were perfused intracardially to fix the brain and for immunohistochemical analysis of c-Fos protein expression within the SNpc. The open field test indicated that SD for 1 or 3 h did not modify motor behavior. However, c-Fos protein expression was increased after 1 h of SD compared with the NSD and 3-h SD groups. These immunohistochemistry data indicate that these periods of SD are not able to produce dopaminergic supersensitivity. Nevertheless, the increased expression of c-Fos within the SNpcsuggests that dopaminergic nigral activation was triggered by SD earlier than motor responsiveness. Dopamine-depleted mice(experiment 2) exhibited a similar increase of c-Fos expression compared to control animals indicating that dopamine neurons are still activated in the 1-h SD group despite the exhaustion of dopamine. This finding suggests that this range (2-5-fold) of neuronal activation may serve as a marker of SD.