Atrial Naturietic Peptide (ANP) is a neuropeptide that regulates function of the hypothalamic-pituitary-adrenal (HPA) axis, immune and neuroimmune system, and epigenetic factors. Research has indicated that ANP may mediate alcohol intake, withdrawal, and craving like behaviors. ANP receptors are present in the mesocorticolimbic (MCL) reward pathway of the brain, which includes the nucleus accumbens (Acb) and the ventral tegmental area (VTA). The objectives of the present study were to examine the effects of ANP microinjected into Acb subregions (Shell (Sh), Core (Co), ventral to AcbSh) on operant ethanol (EtOH) self-administration and into posterior VTA (pVTA) on EtOH-seeking behavior of female alcohol-preferring (P) rats. In the first experiment, ANP (0, 10 μg, or 100 μg) was microinjected into subregions of the Acb to determine its effects on EtOH self-administration. In the second experiment, ANP was microinjected into pVTA to determine its effects on Pavlovian Spontaneous Recovery (PSR) of responding, a measure of context-induced EtOH-seeking behavior. Administration of ANP directly into the AcbSh significantly reduced EtOH self-administration compared to vehicle, whereas ANP into the AcbCo or areas directly ventral to the AcbSh did not alter responding for EtOH. Microinjection of ANP into the pVTA significantly reduced responding on the EtOH-associated lever during the PSR test. The data indicate that activation of ANP systems in the (a) AcbSh can inhibit EtOH intake, and (b) in the pVTA can inhibit EtOH-seeking behavior. The results suggest that manipulations of the ANP system could be a potential target for pharmacotherapeutic intervention to treat alcohol use disorder. Supported in part by AA07462, AA07611, AA10717, AA10721, AA013522, AA019366, AA020908, AA022287, and AA024612.