Kvbeta2 binds to K(+) channel alpha subunits from at least two different families (Kv1 and Kv4) and is a member of the aldo-ketoreductase (AKR) superfamily. Proposed functions for this protein in vivo include a chaperone-like role in Kv1 alpha subunit biogenesis and catalytic activity as an AKR oxidoreductase. To investigate the in vivo function of Kvbeta2, Kvbeta2-null and point mutant (Y90F) mice were generated through gene targeting in embryonic stem cells. In Kvbeta2-null mice, Kv1.1 and Kv1.2 localize normally in cerebellar basket cell terminals and the juxtaparanodal region of myelinated nerves. Moreover, normal glycosylation patterns are observed for Kv1.1 and Kv1.2 in whole brain lysates. Thus, loss of the chaperone-like activity does not appear to account for the phenotype of Kvbeta2-null mice, which include reduced life spans, occasional seizures, and cold swim-induced tremors similar to that observed in Kv1.1-null mice. Mice expressing Kvbeta2, mutated at a site (Y90F) that abolishes AKR-like catalytic activity in other family members, have no overt phenotype. We conclude that Kvbeta2 contributes to regulation of excitability in vivo, although not directly through either chaperone-like or typical AKR catalytic activity. Rather, Kvbeta2 relies upon as yet unidentified mechanisms in the regulation of K(+) channel and/or oxidoreductive functions.