Various extracts of brain were prepared with dilute and concentrated acid, acetone and n-butanol. When the extracts were applied to the guinea-pig isolated ileum, they produced a slow contraction which was not prevented by an antihistamine; in addition, the acid and acetone extracts inhibited the action of histamine. Histamine in extracts of brain was separated from other pharmacologically active substances by chromatography on a carboxylic-acid resin and estimated biologically. The bulk of this histamine was found in small particulate material, whereas slow-reacting substance was found in particulate material of greater density. 5-Hydroxytryptamine was in both the small and large particles. There was no relationship between the distribution of these substances and that of succinic dehydrogenase activity. The measurement of histamine in brain both by biological assay on the guinea-pig ileum and by chemical assay using a fluorimetric procedure gave mean values of 53 and 246 ng/g of wet tissue, respectively. The high values obtained by the chemical assay are attributable in part to substances other than histamine which become fluorescent after reaction with o-phthalaldehyde in this procedure.The regional distribution of histamine in brain is similar to that of 5-hydroxytryptamine and noradrenaline (Adam, 1961). The purpose of the present experiments was to see whether or not histamine occurs in particulate material of brain; this material contains most of the 5-hydroxytryptamine, acetylcholine (Whittaker, 1959) and noradrenaline (Chrugciel, 1960) in brain. Accordingly, the subcellular distribution of histamine has been compared with that of 5-hydroxytryptamine. In addition, the subcellular distribution in brain of a slow-reacting substance is described, together with some of the characteristics of the substance; this part of the work arose from difficulties encountered in the separation of histamine from the slow-reacting substances. Finally, the fluorimetric and biological methods for measuring histamine in brain are compared. A preliminary report of this work has appeared (Green,
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