The Rac1 and Rac3 GTPases are co-expressed in the developing nervous system, where they are involved in different aspects of neuronal development, including the formation of synapses. The deletion of both Rac genes determines a stronger reduction of dendritic spines in vitro compared to the knockout of either gene, indicating that Rac1 and Rac3 play a synergistic role in the formation of these structures. Here, we have addressed the role of each GTPase in the formation of dendritic spines by overexpressing either Rac1 or Rac3 in wildtype neurons, or by re-expressing either GTPase in double knockout hippocampal cultures. We show that the Rac3 protein is expressed with Rac1 in developing hippocampal neurons. Overexpression of either GTPase in WT neurons increases the density of dendritic spines, suggesting the involvement of both GTPases in their formation. We also found that the re-expression of either Rac1 or Rac3 in double knockout neurons is sufficient to restore spinogenesis. Rac1 is significantly more efficient than Rac3 in restoring the formation of spines. On the other hand the quantitative analysis in neurons overexpressing or re-expressing either GTPase shows that Rac3 induces a more pronounced increase in the size of the spines compared to Rac1. These enlarged spines form morphological synapses identified by the juxtaposition of postsynaptic and presynaptic markers. Thus, while Rac1 appears more efficient in inducing the formation of mature spines, Rac3 is more efficient in promoting their enlargement. Our study highlights specific roles of Rac1 and Rac3, which may be functionally relevant also to synaptic plasticity.