Elimination of the Kv1.3 voltage-dependent potassium channel gene produces striking changes in the function of the olfactory bulb, raising the possibility that this channel also influences other sensory systems. We have examined the cellular and subcellular localization of Kv1.3 in the medial nucleus of the trapezoid body (MNTB) in the auditory brainstem, a nucleus in which neurons fire at high rates with high temporal precision. A clear gradient of Kv1.3 immunostaining along the lateral to medial tonotopic axis of the MNTB was detected. Highest levels were found in the lateral region of the MNTB, which corresponds to neurons that respond selectively to low-frequency auditory stimuli. Previous studies have demonstrated that MNTB neurons and their afferent inputs from the cochlear nucleus express three other members of the Kv1 family, Kv1.1, Kv1.2, and Kv1.6. Nevertheless, confocal microscopy of MNTB sections coimmunostained for Kv1.3 with these subunits revealed that the distribution of Kv1.3 differed significantly from other Kv1 family subunits. In particular, no axonal staining of Kv1.3 was detected, and most prominent labeling was in structures surrounding the somata of the principal neurons, suggesting specific localization to the large calyx of Held presynaptic endings that envelop the principal cells. The presence of Kv1.3 in presynaptic terminals was confirmed by coimmunolocalization with the synaptic markers synaptophysin, syntaxin, and synaptotagmin and by immunogold electron microscopy. Kv1.3 immunogold particles in the terminals were arrayed along the plasma membrane and on internal vesicular structures. To confirm these patterns of staining, we carried out immunolabeling on sections from Kv1.3(-/-) mice. No immunoreactivity could be detected in Kv1.3(-/-) mice either at the light level or in immunogold experiments. The finding of a tonotopic gradient in presynaptic terminals suggests that Kv1.3 may regulate neurotransmitter release differentially in neurons that respond to different frequencies of sound.