Rapid eye movement sleep deprivation (REMSD) is a potent stressor in rats. Behavioral abnormalities such as passive and active avoidance, locomotor activity, problem solving, sensory information processing, and the development of adaptive copping strategy in response to repeated stress are among the earliest obvious symptoms of REMSD, the mechanism for which remain largely unknown. The aim of this study was to determine whether 96 h of REMSD causes changes in monoamine neurotransmitters concentrations in rat forebrain regions (frontal cortex, FC; parietal cortex, PC, and striatum) that are involved in mediating higher brain functions such as attentional mechanisms, sensory information processing, and locomotor activity, which are severely affected in REMSD conditions. Rats were subjected to 96 h of REMSD using inverted flower pot water tank technique. To account for the stress associated with water tanks, a tank control group (TC) was included where the animals could reside comfortably on a large pedestal in the water tank. Regional brain concentrations of norepinephrine (NE), dopamine (DA), dihydroxyphenyacetic acid (DOPAC), L-3,4-dihydroxyphenylalanine (L-DOPA), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (HIAA) were determined by electrochemical detection using high-performance liquid chromatography. The concentrations of serotonin and its metabolite, HIAA, was reduced in the frontal and parietal cortexes of REMSD rats compared with TC or cage control (CC) group. NE, DA, DOPAC, and HVA concentrations in FC and PC of REMSD animals were remained unchanged compared with TC or CC rats. A significant increase in the concentrations of DA metabolites was observed in the striatum of REMSD rats when compared with CC and TC rats. There was a 29 and 31% increase in the concentration of striatal DA in REMSD group compared to the TC and CC groups, respectively; however, these percentages were not statistically different. Striatal NE, 5-HT, and HIAA concentrations were not significantly different among the three groups. These results suggest that 96 h of REMSD alters dopaminergic and serotonergic systems in different locations in rat brain. The effect of REMSD on the serotonergic systems are localized in the cerebral cortex, whereas dopaminergic metabolism is increased in the striatum.