This work is centered on the involvement of glutamate in spreading depression. Chick eye cup preparations bathed with Ringer solution at 28 degrees C were used. For chemical stimulation KCl was increased and glutamate was added to the Ringer solution. The occurrence of spreading depression was detected by visual observation of the optical changes accompanying the phenomenon. Enzymatic method based on the fluorescence of the reduced form of nicotinamide adenine dinucleotide was used for glutamate dosage in the Ringer's samples. The investigations proceeded under six different experimental conditions, and the results were analysed by nonparametric statistical methods. No correlation was found between spreading depression and glutamate outflux. The glutamate hypothesis to account for spreading depression was discussed, and experimental situations involving glutamate, K+ and Cl- were indicated that proved unfavorable to the incidence of the reaction.