There is increasing evidence for bidirectional communication between glial cells and neurons. In this study, calcium signaling in primary glia/neuron cultures was investigated using video fluorescence imaging and fura-2. Glial cells in culture without neurons showed occasional spontaneous intracellular Ca2+ oscillations but not intercellular Ca2+ waves. By contrast, glial cells in culture with neurons showed frequent spontaneous Ca2+ oscillations as well as propagated intercellular Ca2+ waves. These spontaneous glial intercellular Ca2+ waves often emanated from sites of contact with neurons, but were only occasionally associated with increases in neuronal Ca2+. Mechanical stimulation of a single glial cell induced a glial intercellular Ca2+ wave which was similar in its temporal and spatial characteristics to spontaneous glial Ca2+ waves. Mechanically induced glial Ca2+ waves, but not spontaneous Ca2+ waves, evoked a transient increase in [Ca2+]i or a change in the pattern of spontaneous Ca2+ oscillations in a small percentage (< 10%) of neighboring neurons. Mechanical stimulation of a single neuron consistently evoked an intercellular Ca2+ wave in neighboring glial cells. These results suggest distinct mechanisms for direct glial-neuronal and neuronal-glial communication. These signaling pathways may play important roles in both function and pathology in the central nervous system.