The Kv3.1 potassium channel can be distinguished from most other delayed rectifier channels by its very high threshold of activation and lack of use-dependent inactivation. This allows neurons that express this channel to fire at very high frequencies. We have now found that this feature of the Kv3.1 channel is strongly influenced by its constitutive phosphorylation by the enzyme casein kinase II. Using stably transfected Chinese hamster ovary cells expressing Kv3.1, we show that Kv3.1 is highly phosphorylated under basal conditions. Whole-cell patch clamp recordings were used to characterize the electrophysiological consequence of dephosphorylation using alkaline phosphatase. This enzyme produced an increase in whole-cell conductance and shifted the voltage dependence of activation to more negative potentials by >20 mV. In addition, a similar shift in the voltage dependence of inactivation was observed. These findings were also confirmed in native Kv3.1 channels expressed in medial nucleus of the trapezoid body (MNTB) neurons. Furthermore, inhibitors of casein kinase 2 mimicked the effect of phosphatase treatment on voltage-dependent activation and inactivation, whereas inhibitors of protein kinase C failed to alter these parameters. The combination of biochemical and electrophysiological evidence suggests that the biophysical characteristics of Kv3.1 that are important to its role in MNTB neurons, allowing them to follow high-frequency stimuli with fidelity, are largely determined by phosphorylation of the channel.