The action of serotonin on medial pontine reticular formation neurons was examined using intracellular electrophysiological methods in rat brainstem slices in vitro. A hyperpolarization associated with a decrease in input resistance was elicited by serotonin in 34% of the neurons, and a depolarization associated with an increase in input resistance was produced in 56% of the neurons. Both responses persisted in the presence of tetrodotoxin. The hyperpolarization resulted from a steady-state increase in outward current which varied with the external potassium concentration in a manner consistent with a conductance increase primarily to this ion. This response was mimicked by the serotonin1 agonist, 5-carboxamidotryptamine, as well as by the serotonin1a agonist, 8-hydroxy-dipropyl aminotetralin hydrobromide, and was blocked by spiperone, an antagonist of serotonin1 sites. The depolarization resulted from a steady-state decrease in outward current which varied with external potassium. The depolarization was mimicked by the serotonin2 agonist, alpha-methyl-5-hydroxytryptamine, and was blocked by the serotonin2 antagonist, ketanserin. Neither of these agents had any effect upon serotonin-induced hyperpolarizations. In conclusion, the excitability of medial pontine reticular formation neurons is influenced by serotonin acting to increase or decrease potassium conductance(s). These opposing effects reflect actions on distinct serotonin receptor subtypes that are segregated to distinct populations of medial pontine reticular formation neurons.