Despite several overlapping functions of cannabinoid receptor 1 (CB1 receptor), somatostatin (SST), and neuronal nitric oxide synthase (nNOS) in the hypothalamus, nothing is currently known whether CB1 receptor-positive neurons coexpress SST and nNOS. In the present study, we describe the colocalization of CB1 receptor with SST and nNOS in the rat brain hypothalamus. In the hypothalamus, the distributional patterns and colocalization of CB1 receptor with SST and nNOS were selective and region specific. CB1 receptor and SST exhibited comparable colocalization (<60%) in paraventricular nucleus (PVN) and periventricular nucleus (PeVN), followed by 20% colocalization in ventromedial hypothalamic nucleus (VMH). Neurons showing colocalization between CB1 receptor and nNOS in PeVN constituted >80%, followed by 60 and 30% in PVN and VMH, respectively. In contrast, SST- and nNOS-positive neurons displayed comparable colocalization (>55%) in PeVN and VMH, followed by PVN (~20%). In the median eminence, CB1 receptor-, SST-, and nNOS-like immunoreactivity was mostly confined to the nerve fibers. The morphological colocalization of CB1 receptor with SST and nNOS shed new light on the understanding of their roles in regulation of physiological and pharmacological response to certain stimuli in hypothalamic nuclei specifically in food intake and energy balance.