The axon initial segment (AIS) is a specialized region in neurons where action potentials are initiated. It is commonly assumed that this process requires a high density of voltage-gated sodium (Na(+)) channels. Paradoxically, the results of patch-clamp studies suggest that the Na(+) channel density at the AIS is similar to that at the soma and proximal dendrites. Here we provide data obtained by antibody staining, whole-cell voltage-clamp and Na(+) imaging, together with modeling, which indicate that the Na(+) channel density at the AIS of cortical pyramidal neurons is approximately 50 times that in the proximal dendrites. Anchoring of Na(+) channels to the cytoskeleton can explain this discrepancy, as disruption of the actin cytoskeleton increased the Na(+) current measured in patches from the AIS. Computational models required a high Na(+) channel density (approximately 2,500 pS microm(-2)) at the AIS to account for observations on action potential generation and backpropagation. In conclusion, action potential generation requires a high Na(+) channel density at the AIS, which is maintained by tight anchoring to the actin cytoskeleton.