This study evaluated the role of excitatory amino acid (EAA) receptor activation in spreading depression (SD), using the in vitro turtle cerebellum as a model system. SD was triggered by electrical stimulation or by elevated K⁺ after the cerebellum had been conditioned for at least 30 min with physiological saline in which most of the chloride had been replaced by propionate. SD was recognized as a transient (1-3 min) negative shift of extracellular potential accompanied by depression of evoked potentials (15-30 min) and an increase of extracellular K⁺ up to 60 mM, which spread across the cerebellum at rates of 1-7 mm/min. SD usually commenced in the granular layer, which apparently contains the 3 major EAA receptor subtypes, quisqualate, kainate and N-methyl-d-aspartate (NMDA), then subsequently spread to the molecular layer, which is largely free of NMDA receptors. Glutamate, aspartate, NMDA, kainate and quisqualate all triggered SD. Kynurenic acid and 2-aminophosphonovaleric acid (APV) inhibited SD under certain conditions further suggesting involvement of EAA receptors. The initiation of SD was blocked by high Mg²⁺ and facilitated in low extracellular Mg²⁺, which also eliminated the delay in molecular layer SD onset. Our data suggest that no one EAA receptor subtype is singly responsible for SD.