Developmental regulation of voltage-dependent delayed rectifier potassium current (I(Kv)) of Xenopus primary spinal neurons regulates the waveform of the action potential. I(Kv) undergoes a tripling in density and acceleration of it activation kinetics during the initial day of its appearance. Another voltage-dependent potassium current, the A current, is acquired during the subsequent day and contributes to further shortening of the impulse duration. To decipher the molecular mechanisms underlying this functional differentiation, we are identifying potassium channel genes expressed in the embryonic amphibian nervous system. Potassium channels consist of pore-forming (alpha) as well as auxiliary (beta) subunits. Here, we report the primary sequence, developmental localization, and functional properties of two Xenopus Kvbeta genes. On the basis of primary sequence, one of these (xKvbeta2) is highly conserved with Kvbeta2 genes identified in other species, whereas the other (xKvbeta4) appears to identify a new member of the Kvbeta family. Both are expressed in developing spinal neurons during the period of impulse maturation but in different neuronal populations. In a heterologous system, coexpression of xKvbeta subunits modulates properties of potassium current that are developmentally regulated in the endogenous I(Kv). Consistent with xKvbeta4's unique primary sequence, the repertoire of functional effects it has on coexpressed Kv1alpha subunits is novel. Taken together, the results implicate auxiliary subunits in regulation of potassium current function and action potential waveforms in subpopulations of embryonic primary spinal neurons.