Mechanical stimulation of adult human and rat pia-arachnoid cell cultures (loaded with calcium indicator dye) produced an increase in calcium in the stimulated cell. This change then propagated rapidly among neighboring cells, producing a calcium wave with a maximum distance of propagation and velocity resembling calcium waves in astrocytes. The pia-arachnoid waves were blocked by either octanol or apyrase, suggesting that propagation might occur either by gap junction communication or extracellular movement of ATP. Calcium waves in pia-arachnoid cells could invade contiguous astrocytes, and vice versa. Gap junction coupling between pia-arachnoid cells and astrocytes was shown by dye transfer experiments, in conjunction with immunostaining for connexin43. We infer that calcium signals from cells in the cortical parenchyma may be transmitted to the pia-arachnoid and might then serve in the induction of neurovascular changes, including those postulated to be responsible for the pain of migraine headache.