People are very likely to start psychoactive drug use during adolescence, an earlier onset being associated with a higher risk of developing addiction later in life. In experiment I, Pre- (postnatal day (pnd) 23-35), Mid- (pnd 36-48), or Post- (pnd 49-61) adolescent mice underwent a restricted-drinking period (2 h/day for 12 days), one bottle containing water and the other containing nicotine (10 mg/l) or water. After this period, Mid-adolescents showed prominent exploration and reduced anxiety in the plus-maze. This ontogenetic profile was dampened by nicotine consumption. After 2 months, these mice were tested in a novel environment (30 min/day for 3 days). Locomotor-habituation profiles were specifically disrupted by nicotine consumption during Mid-adolescence, suggesting this age as a critical period. In experiment II, Mid-adolescent (pnd 35-44) and adult (pnd > 70) mice were pretreated with nicotine (0, 0.03, 0.10, 0.30 mg/kg/day for 10 days). Acute nicotine administration had opposite effects on anxiety in adolescents and adults. At 2 months after pretreatment, we measured levels of AMPA GluR2/3 subunits, thought to be involved in the control of addictive behaviors. Nicotine exposure during Mid-adolescence dose-dependently downregulated these subunits in the striatum and hippocampus, but comparable exposure during adulthood had either opposite or no effects. NMDA NR2A/B subunits were affected by nicotine, but without age-related differences. The present data identified a nicotine-vulnerable age window, characterized by long-term disruption of locomotor habituation and downregulation of AMPA receptors. These findings support neurobiological vulnerability to drugs in adolescent humans.