The supply of choline to the brain and the interrelationship between acetylcholine metabolism in the brain, choline in cerebrospinal fluid (CSF) and choline in plasma has been investigated by studying the transport of deuterium labelled choline from plasma toCSF in rabbits. The experiments were performed either by i.v. infusion of labelled choline or by administering deuterium choline in the diet until steady state levels were reached. All assays of labelled as well as unlabelled choline and acetylcholine were carried out by gas chromatographic-mass spectrometric technique. From these experiments it was found that the half-lives of the disappearance of labelled choline in plasma and CSF were similar (15 min) and the mole fraction of labelled choline in plasma was approximately 2.4 times that in CSF. The half-life of labelled acetylcholine in brain was about 25 min. In the time interval 10-20 min, the mole fraction of brain acetylcholine was the same as that of CSF choline. When infusion was carried out during pentobarbital anaesthesia the mole fraction of labelled brain acetylcholine decreased and that of labelled CSF choline increased compared to those of conscious animals. At steady state the mole fraction of labelled choline in plasma was nearly twice as high as that in CSF which in turn was significantly higher than that of acetylcholine in the brain. From these results it is suggested that in rabbit, free and derived choline in plasma are supplied to the brain in equal amounts. Furthermore it is suggested that choline is transported from plasma, via the extracellular space of the brain to CSF. In the extracellular space a part of choline is diverted and exchanged with choline in the acetycholine metabolic pathway.