Nitric oxide (NO) is a neuronal messenger molecule that plays important roles in the development, maintenance, and functional modifications of brain circuits. We investigated whether the NO levels at different postnatal day (P) periods of the brain develop interference with the locomotion in a novel environment during the postpuberal age (P60). First, using the determination of the nitrite accumulation, we evaluated whether treatment with the NO-synthase inhibitor N-nitro-L-arginine (L-NNA) during different neonatal ages (P1 to P3, P4 to P6, and P7 to P9) affected the levels of NO activity in different regions in the neonatal brain of the rat. We then evaluated whether the locomotor activity in the adult rat (P60) is affected by the blocking of the neonatal NO-activity during a specific period of the development of the nervous system. Neonatal rats with L-NNA administration at P4 to P6 and P7 to P9 show a significant decrease in the levels of NO activity in all the brain regions. However, the blocking of NO synthesis during the neonatal period between P4 to P6 produced an increase in the locomotion after puberty. These data suggest that during a specific step in the development of the brain, the NO levels may play a critical role in the structures that control the spontaneous locomotion in a novel environment after puberty.