Significant amounts of glucagon immunoreactivity (GI) and glucagon-like immunoreactivity (GLI) were found in two portions of the canine brain-the hypothalamus and brain stem. Ratios of GLI to GI were low in the hypothalamus, and variable but low in the brain stem. Dilution curves of the extracts from both regions showed similar patterns to that of glucagon in radioimmunoassays using specific (30K) and cross-reacting (YG9) antisera. The hypothalamic extract contained a larger amount of 3, 500 MW fraction comparable to glucagon and a lesser amount of 9, 000 MW fraction ressembling proglucagon. While displacement of 125I-glucagon from the rat liver cell membrane by the main fraction of the hypothalamus was compatible with that of glucagon, activation of adenylate cyclase by the hypothalamus extract was higher than glucagon and that by the extract of the brain stem was lower than glucagon. Although purificatiou was not sufficient and specific binding of circulating glucagon to the brain has not been overlooked, the hypothalamic extract at least contains material immunologically and probably biologically resembling glucagon.