Nicotine administration induces hypophagia. Because of the involvement of hypothalamic neurotransmitters in food intake control, we hypothesized that increased activity of the lateral hypothalamic dopamine (LHA-DA) and/or serotonin (LHA-5-HT) may be responsible for nicotine-induced hypophagia. Either 4 mM nicotine or vehicle was administered via reverse microdialysis technique into the LHA of overnight food-deprived rats for 60 min; then food was provided for 40 min. The LHA-DA, 5-HT and their intermediate metabolites, DOPAC and 5-HIAA, were continuously measured during 20-min intervals before, during, and after nicotine administration. Continuous nicotine administration for 60 min increased LHA-DA and DOPAC concentrations during the first 40 min, and induced a long-lasting increase in LHA-5-HT release, until 120 min after the start nicotine administration, even when nicotine administration was stopped. The food intake during the 40-min refeeding period was significantly lower when rats received nicotine. Eating induced a significant and short-lasting increase in the LHA-DA and a long-lasting increase in the LHA-5-HT. These findings indicate that nicotine enhances dopaminergic and serotonergic activity in the LHA, and that the enhanced LHA-5-HT activity may contribute to nicotine-induced hypophagia.