Synapse plasticity, in particular, formation of new synapses, plays crucial roles in learning and memory. We have developed a convenient assay system for measuring the number of newly formed synapses between cultured rat cerebrocortical neurons using the multisite fluorometry system of intracellular calcium. We found that cultured neurons exhibited spontaneous oscillatory changes in intracellular calcium levels and that the frequency of the oscillation was strongly correlated with synaptic density. Combined with immunohistochemical studies, this assay system enables us to study the molecular mechanism of synapse formation, in particular, the involvement of ecto-protein kinase. Other applications of the assay system are discussed here.