Dendritic spines mediate most excitatory transmission in the mammalian CNS and have been traditionally considered stable structures. Following the suggestion that spines may ‘twitch’, it has been recently shown that spines are capable of rapid morphological rearrangements. Because of the role of the small GTPases from the Rho family in controlling neuronal morphogenesis, we investigated the effects of several members of this biochemical signaling pathway in the maintenance of the morphology of extant dendritic spines by combining biolistic transfection of pyramidal neurons in cultured cortical and hippocampal slices with two-photon microscopy. We find a variety of effects on the density and morphology of dendritic spines by expressing either constitutively active or dominant negative forms of several small GTPases of the Rho family, by blocking the entire pathway with Clostridium difficile toxin B or by blocking Rho with C3 transferase. We propose a model where Rac promotes spine formation, while Rho prevents it. We conclude that the small GTPases provide antagonistic control mechanisms of spine maintenance in pyramidal neurons.