Kv1 potassium channels consist of pore-forming alpha subunits as well as auxiliary beta subunits. In heterologous systems, Kv1alpha subunits suffice for induction of voltage-dependent potassium current (I(Kv)). Although Kv1 channels can be expressed without auxiliary subunits in heterologous systems, coexpression with Kvbeta subunits has dramatic effects on surface expression and kinetic properties. Much less is known about the functional roles of Kvbeta subunits in vivo, despite their presence in the majority of native Kv1 channel complexes. We used an antisense approach to probe the contribution of Kvbeta2 subunits to native Kv1 channel function in embryonic myocytes. We compared the effects of antisense Kvbeta2 treatment on the whole cell I(Kv) to those produced by overexpression of a dominant-negative Kv1alpha subunit. The reductions in the maximal potassium conductance produced by antisense Kvbeta2 treatment and elimination of Kv1alpha subunit function were not significantly different from each other. In addition, simultaneous elimination of Kv1alpha and Kvbeta2 subunit function resulted in no further reduction of the maximal conductance. The Kv channel complexes targeted by Kvbeta2 and/or Kv1alpha subunit elimination contributed to action potential repolarization because elimination of either or both subunits led to increases in the duration of the action potential. As for potassium conductance, the effects of elimination of both alpha and beta subunits on the duration of the action potential were not additive. Taken together, the results suggest that Kv1 potassium channel complexes in vivo have a strong requirement for both alpha and beta subunits.