In fast-spiking neurons such as those in the medial nucleus of the trapezoid body (MNTB)1 in the auditory brainstem, Kv3.1 potassium channels are required for high-frequency firing. The Kv3.1b splice variant of this channel predominates in the mature nervous system, and is a substrate for phosphorylation by protein kinase C (PKC) at Ser 503. In resting neurons, basal phosphorylation at this site decreases Kv3.1 current, reducing neuronal ability to follow high-frequency stimulation. We used a phospho-specific antibody to determine which PKC isozymes control serine 503 phosphorylation in Kv3.1b-tranfected cells and in auditory neurons in brainstem slices. Using isozyme-specific inhibitors, we found that the novel PKC-d isozyme, together with the novel PKC-e and conventional PKCs contributed to the basal phosphorylation of Kv3.1b in MNTB neurons. In contrast, only PKC-e and conventional PKCs mediate increases in phosphorylation produced by pharmacological activation of PKC in MNTB neurons or by metabotropic glutamate receptor activation in Kv3.1/mGluR1 co-transfected cells. We also measured the time course of dephosphorylation and recovery of basal phosphorylation of Kv3.1b following brief high-frequency electrical stimulation of the trapezoid body and determined that the recovery process is mediated by both novel PKC-d and PKC-e isozymes and by conventional PKCs. The association between Kv3.1b and PKC isozymes was confirmed by reciprocal co-immunoprecipitation of Kv3.1b with multiple PKC isozymes. Our results suggest that the Kv3.1b channel is regulated by both conventional and novel PKC isozymes and that novel PKC-d contributes specifically to the maintenance of basal phosphorylation in auditory neurons.