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The effects of glyphosate oral exposure (35, 75, 150 and 800 mg/kg bw, 6 days) on brain region monoamine levels of male Wistar rats were examined. Glyphosate-treated rats (35, 75, 150 and 800 mg/kg bw, 6 days), had no visible injury, i.e., no clinical signs of dysfunction were observed. After last dose of glyphosate, serotonin (5-HT), dopamine (DA) and norepinephrine (NE) and its metabolites levels were determined in the brain regions striatum, hippocampus, prefrontal, cortex, hypothalamus and midbrain, by HPLC. Glyphosate caused statistically significant changes in the 5-HT and its metabolite 5-hydroxy-3-indolacetic acid (5-HIAA), DA and its metabolites 3,4-hydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and NE and its metabolite 3-metoxy-4-hydroxyphenylethyleneglycol (MHPG) levels in a brain regional- and dose-related manner. Moreover, glyphosate, dose-dependent, evoked a statistically significant increase in 5-HT turnover in striatum and hypothalamus and in DA turnover in prefrontal cortex and hippocampus, and a statistically significant decrease in NE turnover in prefrontal cortex and hypothalamus. The present findings indicate that glyphosate significantly altered central nervous system (CNS) monoaminergic neurotransmitters in a brain regional- and dose-related manner, effects that may contribute to the overall spectrum of neurotoxicity caused by this herbicide.Glyphosate oral exposure caused neurotoxicity in rats.Brain regions were susceptible to changes in CNS monoamine levels.Glyphosate reduced 5-HT, DA, NE levels in a brain regional- and dose-related manner.Glyphosate altered the serotoninergic, dopaminergic and noradrenergic systems.