The aim of the present study is to examine the effects of serotonin synthesis inhibition with p-Chlorophenylalanine (p-CPA) in rats on (1) anxiety behavior examined in the light-dark crossing test and, (2) regional brain concentration of monoamines (NA, DA and 5-HT) and their metabolites (MHPG, DOPAC, HVA and 5-HIAA) as well as GABA in the hypothalamus, amygdala, hippocampus, midbrain central gray matter and the frontal cortex. Treatment of animals with p-CPA produced a significant increase in time out from the illuminated part of the chamber and in time of locomotor activity in the illuminated part of the chamber. HPLC analysis showed a significant reduction of 5-HT and 5-HIAA concentration in all examined brain regions with the exception of the frontal cortex. Additionally, a significant decrease in DA and its metabolites, DOPAC and HVA occurred in the hypothalamus and amygdala. Moreover, we observed a significant decrease in frontal cortex NA concentration after p-CPA administration. The results of our study suggest that administration of p-CPA is effective in reduction of anxiety through depletion of 5-HT accompanied by diminution of catecholamines, especially DA and its metabolites in the main emotional brain regions.