The lateral superior olive (LSO) is a binaural auditory brain stem nucleus that plays a central role in sound localization. Survival and maturation of developing LSO neurons critically depend on intracellular calcium signaling. Here we investigated the mechanisms by which glutamatergic afferents from the cochlear nucleus increase intracellular calcium concentration in LSO neurons. Using fura-2 calcium imaging in slices prepared from neonatal mice, we found that cochlear nucleus afferents can activate all major classes of ionotropic and metabotropic glutamate receptors, each of which contributes to an increase in intracellular calcium. The specific activation of different glutamate receptor classes was dependent on response amplitudes and afferent stimulus patterns. Low-amplitude responses elicited by single stimuli were entirely mediated by calcium-impermeable AMPA/kainate receptors that activated voltage-gated calcium channels. Larger-amplitude responses elicited by either single stimuli or stimulus trains resulted in additional calcium influx through N-methyl-d-aspartate receptors. Finally, high-frequency stimulation also recruited group I and group II metabotropic glutamate receptors, both of which mobilized intracellular calcium. This calcium release in turn activated a strong influx of extracellular calcium through a membrane calcium channel that is distinct from voltage-gated calcium channels. Together, these results indicate that before hearing onset, distinct patterns of afferent activity generate qualitatively distinct types of calcium responses, which likely serve in guiding different aspects of LSO development.