The subfornical organ (SFO) is one of the circumventricular organs (CVOs) of the mammalian nervous system responsible for maintaining the energy and water and sodium balance. Despite notable interest in the SFO and its physiological functions, the organization of individual populations of SFO cells, as well as their interactions, remain unclearly established. In this study, GABA and nitroxidergic systems of SFO using immunohistochemical (IHC) methods were examined. The brain of male Wistar rats at different stages of postnatal development—postnatal day 7 (P7), 14 (P14), and adult (4–6 months)—was examined. The obtained data allowed the authors to characterize changes in the activity of the GABA- and nitroxidergic systems of the SFO during development. In adult rats, three subpopulations of nitroxidergic cells, differing in the intensity of the reaction and tissue localization, can be distinguished. The revealed morphological heterogeneity of nitroxidergic cells in SFO may reflect their diverse functional status.