Whereas Kv2 subunits modulate potassium current properties carried by Kv1 channel complexes in heterologous systems, little is known about the contributions of Kv2 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 Kv2 subunits in modulation of voltage-dependent potassium current (I_Kv). We focused on (1) two different populations of dorsal spinal neurons that express both Kv2 and Kv1 -subunit genes, and (2) the 24 and 48 hr developmental period, during which I_Kv undergoes developmental regulation. At both 24 and 48 hr, antisense methods produced efficient knock-down of both Kv2 protein and I_Kv. At both times, dominant negative suppression of Kv1 channels also eliminated I_Kv, indicating that Kv1 channels require Kv2 subunits in order to function in dorsal spinal neurons. Even though Kv1 channels determined the I_Kvs of both dorsal neuron types, comparisons of their I_Kv properties revealed important differences at both developmental stages. The latter results support the notion that different Kv1 -subunits and/or post-translational modifications underlie the I_Kvs of the two dorsal neuron types. Overall, the results demonstrate that Kv2 subunits function in vivo as obligatory subunits of Kv1 channels in at least two neuron types and two different developmental stages.