Spreading depression (SD) is a slowly propagating depression of cerebral neuronal activity and transmembrane ionic gradients, that arises in response to a variety of noxious stimuli. SD bears a strong resemblance to gap junction-mediated calcium waves among cultured astrocytes. Here, we show that gap junction-mediated intercellular diffusion is necessary for the generation of SD. Waves of SD in the isolated chicken retina were blocked by five different inhibitors of gap junctional coupling, which was assessed by the intercellular transit of Lucifer Yellow (LY). Each of these gap junction blockers inhibited both the migration of SD and the diffusion of LY in a dose-dependent manner. In contrast, glutamate-evoked calcium influx into retinal cells was not affected by these compounds. The results indicate that intercellular coupling through gap junctions is required for SD. Gap junction-mediated communication might therefore constitute an important mechanism in both normative and pathological brain function.