Abstract Altered levels of extracellular serotonin (5-HT) during development cause structural abnormalities in the neural projections of sensory systems. To better understand the potential role of 5-HT in the development of auditory system projections, we examined 5-HT immunoreactivity (IR) in auditory brainstem nuclei of postnatal mice. We previously observed 5-HT-IR in the lateral superior olive (LSO) of wild type mice. In the current study, we used a genetic model (monoamine oxidase-A knockout mouse) in which brain 5-HT levels are abnormally high to improve detection of 5-HT. In the cochlear nucleus of this knockout, 5-HT-IR cell bodies were observed in the dorsal cochlear nucleus (DCN), a primary relay to the inferior colliculus (IC). In the superior olivary complex, 5-HT-IR somata were observed in the LSO, another relay to the IC. Labeled somata were also observed within the IC itself. The 5-HT immunostaining in all 3 regions was transient and was not observed beyond postnatal day 8. These results suggest that 5-HT may play a role in the branching and refinement of DCN and LSO axon collaterals within the IC, as well as IC axon collaterals within the medial geniculate body. The pattern of expression indicates that 5-HT has a developmental role in select populations of neurons of the ascending auditory pathway prior to any influences of sound-evoked activity.