During the same postnatal period of development when their terminal projection patterns in the midbrain are maturing, lateral superior olivary (LSO) neurons are immunoreactive for serotonin (5-HT). As there is no evidence that LSO neurons synthesize 5-HT, it is likely that they accumulate 5-HT via the 5-HT transporter. To determine if the 5-HT transporter is responsible for 5-HT inside postnatal mouse LSO neurons, pups (postnatal ages 5-6) were treated with fluoxetine and LSO neurons examined for 5-HT. We also evaluated whether LSO neurons containing 5-HT expressed the 5-HT transporter. To further rule out any potential synthesis of 5-HT, brainstem sections of mice at postnatal ages when 5-HT staining is the most robust were stained for the rate-limiting enzyme in the synthesis of 5-HT, tryptophan hydroxylase. Fluoxetine treatment reduced or in most cases, completely eliminated the number of neurons in the LSO stained for 5-HT. Postnatal LSO neurons containing 5-HT were immunoreactive for the 5-HT transporter; in older animals in which 5-HT was no longer observed in the LSO neurons, 5-HT transporter expression was similarly absent. Further, LSO neurons in mice at any age did not stain for tryptophan hydroxylase. These results indicate that LSO neurons express the functional 5-HT transporter to internalize 5-HT; this mechanism may serve to regulate extracellular 5-HT levels during maturation of their terminal endings in the inferior colliculus.