Spontaneous intercellular Ca2+ waves were observed in groups of neurons in two different culture preparations: primary mouse cortical neurons and GT1-1 immortalized neurons. Waves of increased intracellular Ca2+ concentration propagated at rates of 100-200 microns/s over as many as 200 cells and were abolished by the removal of extracellular calcium, by nimodipine, by tetrodotoxin, and by the gap junction inhibitor octanol. A sister clone of the GT1 line, GT1-7 neurons, showed no intercellular Ca2+ waves and were found to have a significantly lower level of connexin26 mRNA than the GT1-1 line. Although we cannot definitively rule out a role for synaptic communication, we propose that intercellular Ca2+ waves in cultured neurons are generated by Ca2+ influx caused primarily by the propagation of depolarization via gap junctions. Intercellular Ca2+ signaling via gap junctions may represent an important mechanism for nonsynaptic neuronal signaling.