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Avian mesotocin (MT) is homologous to oxytocin in mammals. Native Thai chickens (Gallus domesticus) strongly express maternal behaviors including incubation and rearing. However, the role of MT during incubation behavior has never been studied. The objective of this study was to determine the physiological function(s) of the MTergic system in incubation behavior in native Thai chickens. The brains were collected from incubating (INC) and nest-deprived (ND) hens at different time points (days 3, 6, 8, 10, 14, 18, and 21; n=6). Immunohistochemistry technique was used to compare the numbers of MT-immunoreactive (-ir) neurons between the INC and ND hens within the Nucleus supraopticus, pars ventralis (SOv), Nucleus preopticus medialis (POM), and Nucleus paraventricularis magnocellularis (PVN). The results revealed that the numbers of MT-ir neurons within the SOv, POM, and PVN remained high during the incubating stage. The number of MT-ir neurons in the SOv was lower than that of the POM and PVN. Disruption of incubation behavior by nest deprivation caused the numbers of MT-ir neurons within the SOv, POM, and PVN to decrease throughout the observation periods. For the first time, this study demonstrates that the MTergic system within the SOv, POM, and PVN may be involved with incubation behavior. In addition, these results further suggest that the MTergic neurons in these nuclei are not only regulated by rearing behavior but also might have a role in the initiation and maintenance of incubation behavior in this tropical species., (Copyright © 2017 Elsevier GmbH. All rights reserved.)