Waves of elevated intracellular free calcium that propagate between neighboring astrocytes are important for the intercellular communication between astrocytes as well as between neurons and astrocytes. However, the mechanisms responsible for the initiation and propagation of astrocytic calcium waves remain unclear. In this study, intercellular calcium waves were evoked by focal photolysis of a caged calcium ionophore (DMNPE-caged Br A23187) in cultured astrocytes from newborn rats. The focal photolysis of the caged compound resulted in the increase in intracellular calcium in a single astrocyte, and this increase then propagated to neighboring astrocytes. We also analyzed the spatiotemporal characteristics of the intercellular calcium waves, and estimated the propagation pathways for them. The method using a caged calcium ionophore described in this study provides a new in vitro model for the analysis of intercellular calcium waves.