Experimental evidence recently obtained suggests that synaptogenesis is a tripartite event in which not only pre- and post-synaptic neurons but also glial cells play a key role. However, the molecular mechanisms by which glia modulate the formation of synapses in the CNS remain poorly understood. In the present study, we analyzed the role of astrocytes in synapse formation in cultured hippocampal rat neurons. For these experiments, hippocampal neurons were cultured in the presence or absence of a monolayer of astrocytes. Our results indicated that hippocampal neurons cultured in the presence of astrocytes formed more synapses than the ones cultured in their absence only when kept in N2 serum-free medium. To get insights into the potential molecular mechanisms underlying this effect, we analyzed the expression of proteins known to induce synapse formation in hippocampal neurons. A significant increase in agrin expression was detected in astrocytes cultured in N2 serum-free medium when compared with the ones cultured in serum containing medium. Experiments performed using different components of the N2 mixture indicated that progesterone induced the expression of agrin in astrocytes. Taken collectively, these results provide evidence supporting a role for astrocytes in synapse formation in central neurons. Furthermore, they identified agrin as a potential mediator of this effect, and astrocytes as a bridge between the endocrine and nervous systems during synaptogenesis.