The brainstem dorsal raphe nucleus (DRN) contains an abundant distribution of nitric oxide (NO) synthase (NOS)-containing neuronal profiles in two distinct populations: faint- and intense-immunoreactive cells in midline (ventromedial and dorsomedial) and lateral wing subregions, respectively. This study tested the hypothesis that different functional dynamics underlie the topography of NOS-containing cells in the DRN rostrocaudal and mediolateral neuraxis by using a capsaicin challenge paradigm (50 mg/kg, subcutaneous). Compared with vehicle, capsaicin significantly and preferentially increased nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d, an index of constitutive NOS) reactivity in the rostral midline and caudal lateral wing subregions. Furthermore, capsaicin activated more Fos-positive cells than vehicle within all subregions of the DRN but with a caudal versus rostral predominance in activation pattern. In addition, a high proportion of capsaicin-induced Fos cells in the midline but almost none in lateral wing stained for NADPH-d. These observations suggest the existence of two functionally distinct populations of NOS neurons in the DRN. Furthermore, capsaicin increased galanin immunoreactivity with predominant staining in cell soma and fiber processes in midline and lateral wing subregions of the nucleus, respectively. The total capsaicin-induced galanin immunoreactivity was higher in rostral versus caudal DRN, and a high proportion of galanin-positive cells in the midline also contained NADPH-d and neuronal NOS, thus suggesting a potential NO-galanin interaction in these neurons. The differential pattern of Fos/NADPH-d colocalization across the nucleus suggests that midline and lateral wing NOS neurons of the DRN express their neuromodulatory actions on discrete efferent targets via different intracellular mechanisms.