Rapid eye movement sleep (REMS) plays important role in maintenance of normal brain functions. Neurons containing various neurotransmitters in different brain regions interact to regulate this complex phenomenon in health and diseases. The number of neuronal projections, their firing rates and neurotransmitter levels vary in different brain regions under various conditions leading to normal or altered patho-physio-behavioral states. In this study using high performance liquid chromatography (HPLC) we quantified noradrenaline (NA) and gamma-amino butyric acid (GABA) levels in locus coeruleus (LC), dorsal raphe (DR), pedunculo-pontine tegmentum (PPT), frontal lobe (FL), cortex and hippocampus (Hippo) in control and after 96 h REMS deprivation (REMSD) rats. Normal free moving control (FMC) rats were taken as standard cage controls. To rule out non-specific effects large platform control (LPC) and post-REMSD recovery (REC) were carried out. The levels of NA and GABA in discrete brain regions upon REMSD were statistically compared with all the controls. Upon REMSD, although NA levels significantly increased and the GABA levels decreased in the LC, PPT and cortex, in Hippo their levels showed opposite responses. Only NA levels increased in FL, while only GABA levels were decreased in the DR after REMSD. Most of the altered neurotransmitter levels returned to normal levels in REC rats. The findings help understanding the neurochemical basis of REMSD and its associated effects.