In rodents, previous findings indicate critical involvement of the medial preoptic area (MPOA) in the neural control of maternal behavior. However, the specification of the particular MPOA subregions involved in maternal behavior and the identification of the neurochemical phenotype(s) of the essential neurons demands additional study. Therefore, we investigated the chemical neuroanatomy of the essential MPOA subregion for maternal behavior in C57BL/6J female mice. Using the oxytocinergic neurons in the dorsal MPOA as a primary regional marker, we first assessed the distribution of c-Fos-expressing neurons in the MPOA during maternal behavior using immunohistochemistry. Results showed that non-oxytocinergic neurons in the dorsal and ventral MPOA prominently expressed c-Fos during maternal behavior. Then using excitotoxic lesion studies, we determined the specific MPOA area that is necessary for maternal behavior. Bilateral lesions of the central MPOA, where c-Fos was expressed only moderately, effectively disrupted maternal behavior, although lesions to the dorsal and ventral MPOA regions were ineffective. These centrally lesioned females were highly infanticidal irrespective of their previous maternal experience. Neurochemical investigations showed that more than 75% of the c-Fos-expressing neurons in central MPOA were GABAergic. Many of them also expressed galanin, neurotensin, and/or tachykinin2 mRNAs. Finally, the central MPOA was populated by numerous glutamatergic neurons, although only a small percentage of these neurons colocalized with c-Fos. To conclude, the central MPOA is the indispensable subregion for mouse maternal behavior, and GABAergic and/or peptidergic neurons in this area were transcriptionally activated during maternal behavior. J. Comp. Neurol. 521:1633–1663, 2013. © 2012 Wiley Periodicals, Inc.