The distal end of the axon initial segment (AIS) is the preferred site for action potential initiation in cortical pyramidal neurons because of its high Na + channel density. However, it is not clear why action potentials are not initiated at the proximal AIS, which has a similarly high Na + channel density. We found that low-threshold Na v 1.6 and high-threshold Na v 1.2 channels preferentially accumulate at the distal and proximal AIS, respectively, and have distinct functions in action potential initiation and backpropagation. Patch-clamp recording from the axon cut end of pyramidal neurons in the rat prefrontal cortex revealed a high density of Na + current and a progressive reduction in the half-activation voltage (up to 14 mV) with increasing distance from the soma at the AIS. Further modeling studies and simultaneous somatic and axonal recordings showed that distal Na v 1.6 promotes action potential initiation, whereas proximal Na v 1.2 promotes its backpropagation to the soma. © 2009 Nature America, Inc.