The Rho family of GTPases have important roles in the morphogenesis of the dendritic spines(1-3) of neurons in the brain and synaptic plasticity(4-9) by modulating the organization of the actin cytoskeleton(10). Here we used two-photon fluorescence lifetime imaging microscopy(11-13) to monitor the activity of two Rho GTPases-RhoA and Cdc42-in single dendritic spines undergoing structural plasticity associated with long-term potentiation in CA1 pyramidal neurons in cultured slices of rat hippocampus. When long-term volume increase was induced in a single spine using two-photon glutamate uncaging(14,15), RhoA and Cdc42 were rapidly activated in the stimulated spine. These activities decayed over about five minutes, and were then followed by a phase of persistent activation lasting more than half an hour. Although active RhoA and Cdc42 were similarly mobile, their activity patterns were different. RhoA activation diffused out of the stimulated spine and spread over about 5 mm along the dendrite. In contrast, Cdc42 activation was restricted to the stimulated spine, and exhibited a steep gradient at the spine necks. Inhibition of the Rho-Rock pathway preferentially inhibited the initial spine growth, whereas the inhibition of the Cdc42-Pak pathway blocked the maintenance of sustained structural plasticity. RhoA and Cdc42 activation depended on Ca2+/calmodulin-dependent kinase (CaMKII). Thus, RhoA and Cdc42 relay transient CaMKII activation(13) to synapse-specific, long-term signalling required for spine structural plasticity.