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Differences in cytoskeleton organization are key determinants of the architecture and dynamics of axons and dendrites. This is most clearly illustrated by the diverse pools of microtubule-associated proteins in axons and dendrites. Whether similar polarized organization occurs for actin regulatory proteins remains to be determined. To address this issue, we analyzed the intracellular distribution of the Rho GTPases, RhoA, Rac1, and cdc42 in hippocampal neurons in culture. We report that all three Rho members are evenly distributed during the time of axon and dendrite sprouting. This is not the case in mature neurons, as RhoA enriches in dendrites, Rac1 in axons, and Cdc42 is equally abundant in both domains. Polarized segregation of the actin regulatory machinery in mature neurons might play an important role in axonal and dendritic architectural plasticity.