Whereas Kvbeta2 subunits modulate potassium current properties carried by Kv1 channel complexes in heterologous systems, little is known about the contributions of Kvbeta2 subunits to native potassium channel function. Using antisense approaches and in situ recordings from Xenopus embryo spinal cord neurons, we tested the in vivo roles of Kvbeta2 subunits in modulation of voltage-dependent potassium current (IKv). We focused on 1) two different populations of dorsal spinal neurons that express both Kvbeta2 and Kv1 alpha-subunit genes and 2) the 24- and 48-h developmental period, during which IKv undergoes developmental regulation. At both 24 and 48 h, antisense methods produced efficient knock-down of both Kvbeta2 protein and IKv. At both times, dominant negative suppression of Kv1 channels also eliminated IKv, indicating that Kv1 channels require Kvbeta2 subunits to function in dorsal spinal neurons. Even though Kv1 channels determined the IKv values of both dorsal neuron types, comparisons of their IKv properties revealed important differences at both developmental stages. The latter results support the notion that different Kv1 alpha-subunits and/or posttranslational modifications underlie the IKv values of the two dorsal neuron types. Overall, the results demonstrate that Kvbeta2 subunits function in vivo as obligatory subunits of Kv1 channels in at least two neuron types and two different developmental stages.