Serotonin (5-hydroxytryptamine, 5-HT) released by platelets, mast cells, and immunocytes is a potent inflammatory mediator which modulates pain and itch sensing in the peripheral nervous system. The serotonergic receptors expressed by primary afferent neurons involved in these sensory functions are not fully identified and appear to be to a large extent species dependent. Moreover, the mechanisms through which 5-HT receptor activation is coupled to changes in neuronal excitability have not been completely revealed. Using a combination of in vitro (calcium and voltage imaging and patch-clamp) and in vivo behavioral methods, we used both male and female Wistar rats to provide evidence for the involvement of two 5-HT receptor subtypes, 5-HT1A and 5-HT3, in mediating the sustained and transient effects, respectively, of 5-HT on rat primary afferent neurons involved in pain and itch processing. In addition, our results are consistent with a model in which sustained serotonergic responses triggered via the 5-HT1A receptor are due to closure of background potassium channels, followed by membrane depolarization and action potentials, during which the activation of voltage-gated calcium channels leads to calcium entry. Our results may provide a better understanding of mammalian serotonergic itch signaling.