AbstractMouse brain cortex slices have been incubated with tritium labelled acetylcholine, choline and atropine. The subcellular distribution of radioactivity has then been studied by homogenization of the slices in sucrose and separation of the subcellular particles on a continuous exponential sucrose gradient. It was found that about 30 per cent of the radioactivity in the slices were localized in the fraction containing nerve‐endings and mitochondria. Using differential centrifugation and gradient centrifugation of the crude mitochondrial fraction obtained, it was found that acetylcholine is specifically bound to the nerve‐ending particles. Choline had a subcellular distribution similar to acetylcholine, while atropine was not specifically bound to the nerve‐en