This study examined the passive membrane and action potential properties of serotonergic and non serotonergic neurons in the rostral ventromedial medulla (RVM) of the rat using whole cell patch clamp recording techniques in the slice. Serotonergic neurons were identified by immunoreactivity for tryptophan hydroxylase (TrpH). Spinally-projecting neurons were retrogradely labeled with with 1' dioactadecyl 3,3,3',3' tetramethylindocarbodyanine perchlorate (DiI). Three types of neurons were identified within both spinally-projecting serotonergic and non-serotonergic populations. Type 1 neurons exhibited irregular or sporadic spontaneous activity interspersed with periods of quiescence. Type 2 neurons were not spontaneously active, and were additionally discriminated by a more negative resting membrane potential and a larger amplitude action potential. Type 3 neurons fired repetitively without pause. Serotonergic neurons had a higher membrane resistance and greater action potential half width than their non serotonergic counterparts, and rarely exhibited a fast after hyperpolarization. Serotonergic Type 3 neurons also fired more slowly and regularly than non serotonergic Type 3 neurons. Comparison of electrophysiological and immunohistochemical characteristics suggested that the smallest Type 3 serotonergic neurons had an increased risk of immunohistochemical "misclassification" due to failure to detect TrpH, possibly due to more complete dialysis of intracellular contents during lengthy recordings. This risk was minimal for Type 1 or Type 2 serotonergic neurons. The three different types of spinally-projecting serotonergic neurons also differed markedly in their responsiveness to the mu opioid receptor agonist [D-Ala², NMePhe⁴, Gly⁵-ol]enkephalin. These results provide important new electrophysiological and pharmacological evidence for a significant heterogeneity among spinally-projecting serotonergic RVM neurons. They may also provide a basis for resolving the controversy concerning the role of serotonergic RVM neurons in opioid analgesia.