The asymmetric distribution of proteins to distinct domains in the plasma membrane is crucial to the function of many polarized cells. In epithelia, distinct apical and basolateral surfaces are maintained by tight junctions that prevent diffusion of proteins and lipids between the two domains. Polarized neurons maintain axonal and somatodendritic plasma membrane domains without an obvious physical barrier. Indeed, the artificial lipid Dil encounters no diffusion barrier at the presumptive domain boundary, the axon hillock. By measuring the lateral mobility of membrane proteins using optical tweezers, we show here that some membrane proteins exhibit markedly reduced mobility in the initial segment of the axon. Disruption of F-actin and low levels of dimethyl sulphoxide (DMSO) abolish this diffusion barrier and lead to redistribution of membrane markers that had previously been polarized. Immobilization in the initial segment may reflect, at least in part, differential tethering to cytoskeletal components. Therefore, the ability to maintain a polarized distribution of membrane proteins depends on a specialized domain at the initial segment of the axon, which restricts lateral mobility and serves as a new type of diffusion barrier that acts in the absence of cell-cell contact.