The prepositus hypoglossi nucleus (PHN) and interstitial nucleus of Cajal (INC) are involved in controlling horizontal and vertical gaze, respectively. Previous studies have shown that PHN neurons exhibit depolarized or hyperpolarized responses to serotonin (5-hydroxytryptamine, 5-HT). However, serotonergic modulation of INC neurons has not been examined. Furthermore, the relationship between 5-HT-induced responses and neuron types based on neurotransmitter phenotypes has not been clarified. In this study, we investigated 5-HT-induced current responses in PHN and INC neurons and the distributions of distinct current responses in different neuron types using whole-cell recordings of wild-type and transgenic rat brainstem slices. Local application of 5-HT to the cell soma confirmed that slow inward (SI) and slow outward (SO) currents were mediated by 5-HT2 and 5-HT1A receptors, respectively. Furthermore, fast inward (FI) currents that were mediated by 5-HT3 receptors were observed. These three current responses were observed in both PHN and INC neurons. Analyses of the distributions of the three current responses revealed that fluorescently identified glutamatergic and inhibitory neurons in the PHN showed high proportions of SI and SO currents, respectively, whereas glutamatergic and inhibitory neurons in the INC showed mainly SO currents. When PHN and INC neurons were characterized based on firing patterns, the proportions of the currents depended on the firing patterns. The different distributions of 5-HT-induced currents suggest distinct serotonergic modulation modes specific to horizontal and vertical gaze control.