Within the developing Xenopus spinal cord, voltage-gated potassium (Kv) channel genes display different expression patterns, many of which occur in opposing dorsal-ventral gradients. Regional differences in Kv gene expression would predict different patterns of potassium current (I(Kv)) regulation. However, during the first 24 h of postmitotic differentiation, all primary spinal neurons undergo a temporally coordinated upregulation of I(Kv) density that shortens the duration of the action potential. Here, we tested whether spinal neurons demonstrate regional differences in I(Kv) regulation subsequent to action potential maturation. We show that two types of neurons, I and II, can be identified in culture on the basis of biophysical and pharmacological properties of I(Kv) and different firing patterns. Chronic increases in extracellular potassium, a signature of high neuronal activity, do not alter excitability properties of either neuron type. However, elevating extracellular potassium acutely after the period of action potential maturation leads to different changes in membrane properties of the two types of neurons. I(Kv) of type I neurons gains sensitivity to the blocker XE991, whereas type II neurons increase I(Kv) density and fire fewer action potentials. Moreover, by recording from neurons in vivo, we found that primary spinal neurons can be identified as either type I or type II. Type I neurons predominate in dorsal regions, whereas type II neurons localize to ventral regions. The findings reveal a dorsal-ventral gradient for I(Kv) regulation and a novel form of neuronal plasticity in spinal cord neurons.