Fast-spiking GABAergic interneurons of the neocortex and hippocampus fire high-frequency trains of brief action potentials with little spike-frequency adaptation. How these striking properties arise is unclear, although recent evidence suggests K(+) channels containing Kv3.1-Kv3.2 proteins play an important role. We investigated the role of these channels in the firing properties of fast-spiking neocortical interneurons from mouse somatosensory cortex using a pharmacological and modeling approach. Low tetraethylammonium (TEA) concentrations (</=1 mM), which block only a few known K(+) channels including Kv3.1-Kv3.2, profoundly impaired action potential repolarization and high-frequency firing. Analysis of the spike trains evoked by steady depolarization revealed that, although TEA had little effect on the initial firing rate, it strongly reduced firing frequency later in the trains. These effects appeared to be specific to Kv3.1 and Kv3.2 channels, because blockade of dendrotoxin-sensitive Kv1 channels and BK Ca(2+)-activated K(+) channels, which also have high TEA sensitivity, produced opposite or no effects. Voltage-clamp experiments confirmed the presence of a Kv3.1-Kv3.2-like current in fast-spiking neurons, but not in other interneurons. Analysis of spike shape changes during the spike trains suggested that Na(+) channel inactivation plays a significant role in the firing-rate slowdown produced by TEA, a conclusion that was supported by computer simulations. These findings indicate that the unique properties of Kv3.1-Kv3.2 channels enable sustained high-frequency firing by facilitating the recovery of Na(+) channel inactivation and by minimizing the duration of the afterhyperpolarization in neocortical interneurons.