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 (Na1) channels. Paradoxically, the results of patch-clamp studies suggest that the Na1 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 Na1 imaging, together with modeling, which indicate that the Na1 channel density at the AIS of cortical pyramidal neurons is ~50 times that in the proximal dendrites. Anchoring of Na1 channels to the cytoskeleton can explain this discrepancy, as disruption of the actin cytoskeleton increased the Na1 current measured in patches from the AIS. Computational models required a high Na1 channel density (~2,500 pS lm–2) at the AIS to account for observations on action potential generation and backpropagation. In conclusion, action potential generation requires a high Na1 channel density at the AIS, which is maintained by tight anchoring to the actin cytoskeleton.