This study combines functional and anatomical characterization of neurons in the rostral ventromedial medulla (RVM) to show distinct neurochemical phenotypes between functional classes of neurons. The RVM contains three functional classes of neurons: off cells show a pause in spontaneous activity prior to a nociceptive withdrawal reflex; on cell activity increases prior to a nociceptive reflex; and neutral cell activity does not change significantly during the nociceptive reflex. We determined if serotonin, glutamate decarboxylase (GAD67), or the kappa opioid receptor (KOR) were differentially located within these cell types as predicted by previous studies. In this study, RVM neurons were recorded extracellularly, functionally characterized, and juxtacellularly labeled with biotinamide. Fixed sections were processed for detection of biotinamide and immunfluorescence either for serotonin or for KOR and GAD67. In the first study, serotonin was found exclusively in a subset of neutral cells (33%). These data substantiate previous findings that serotonin is found in some neutral cells whose role in nociception remains unclear. In the second study, we found KOR immunoreactivity in most off (86%) and neutral (80%) cells but rarely in on (13%) cells. We also found GAD67 immunoreactivity in most off (93%) and neutral cells (80%) but less frequently in on cells (63%). Most KOR-immunoreactive cells (16 of 17) also contained GAD67 immunoreactivity regardless of cell classification. These findings support the hypothesis that KOR agonists directly inhibit off and neutral cell activity. The majority of the off and neutral cells are GABAergic, and some on cells are also GABAergic.