Interactions between astrocytes and endothelial cells are believed to play an important role in the control of blood-brain barrier permeability and transport. Astrocytes and endothelial cells respond to a variety of stimuli with an increase of intracellular free calcium ([Ca2+]i) that is propagated to adjacent cells as an intercellular Ca2+ wave. We hypothesized that intercellular Ca2+ signaling also occurs between astrocytes and endothelial cells, and we investigated this possibility in co-cultures of primary astrocytes and an endothelial cell line using caged messengers. Intercellular Ca2+ waves, induced by mechanical stimulation of a single cell, propagated from astrocytes to endothelial cells and vice versa. Intercellular Ca2+ waves could also be induced by flash photolysis of pressure-injected caged inositol trisphosphate (IP3) and also by applying the flash to remote noninjected cells. Ca2+ waves induced by flash photolysis propagated from endothelial cells to astrocytes but not from astrocytes to endothelial cells even though caged IP3 diffused between the two cell types. Flash photolysis of caged Ca2+ (NP-EGTA) resulted in an increase of [Ca2+]i but did not initiate an intercellular Ca2+ wave. We conclude that an increase of IP3 in a single cell is sufficient to initiate an intercellular Ca2+ wave that is propagated by the diffusion of IP3 to neighboring cells and that can be communicated between astrocytes and endothelial cells in co-culture. By contrast, Ca2+ diffusion via gap junctions does not appear to be sufficient to propagate an intercellular Ca2+ wave. We suggest that intercellular Ca2+ waves may play a role in astrocyte-endothelial interactions at the blood-brain barrier.