The firing pattern of auditory neurons is determined in part by the type of voltage-sensitive potassium channels expressed. The expression patterns for two high-threshold potassium channels, Kv3.1 and Kv3.3, that differ in inactivation properties were examined in the rat auditory system. The positive activation voltage and rapid deactivation kinetics of these channels provide rapid repolarization of action potentials with little effect on action potential threshold. In situ hybridization experiments showed that Kv3.3 mRNA was highly expressed in most auditory neurons in the rat brainstem, whereas Kv3.1 was expressed in a more limited population of auditory neurons. Notably, Kv3.1 mRNA was not expressed in neurons of the medial and lateral superior olive and a subpopulation of neurons in the ventral nucleus of the lateral lemniscus. These results suggest that Kv3.3 channels may be the dominant Kv3 subfamily member expressed in brainstem auditory neurons and that, in some auditory neurons, Kv3.1 and Kv3.3 may coassemble to form functional channels. The localization of Kv3.1 protein was examined immunohistochemically. The distribution of stained somata and neuropil varied across auditory nuclei and correlated with the distribution of Kv3.1 mRNA-expressing neurons and their terminal arborizations, respectively. The intensity of Kv3.1 immunoreactivity varied across the tonotopic map in the medial nucleus of the trapezoid body with neurons responding best to high-frequency tones most intensely labeled. Thus, auditory neurons may vary the types and amount of K(+) channel expression in response to synaptic input to subtly tune their firing properties.