The metabolism of choline by rat brain, plasma, and liver was investigated using combined gas chromatography mass spectrometry following microwave irradiation and treatment with deuterium-labeled choline. Methods were established to measure simultaneously the concentrations of six choline-containing compounds and the incorporation of labeled choline into each of them. Intravenous injection of [2H4]-choline led to initial labeling of choline, acetylcholine, and phosphocholine in rat brain, with all of the label eventually entering the phosphocholine pool. When labeled choline was administered in the diet its rate of incorporation into choline, phosphatidylcholine, and combined choline plasmalogen and lysophosphatidylcholine in the plasma and liver and into choline, acetylcholine, phosphocholine, glycerophosphorylcholine, phosphatidylcholine, and combined choline plasmalogen and lysophosphatidylcholine in the brain were determined. Choline, phosphatidylcholine, and combined choline plasmalogen and lysophosphatidylcholine in the plasma had similar specific activities. In the cortex and the striatum, choline and combined choline plasmalogen and lysophosphatidylcholine fraction generally had the highest specific activities. The time course of the post-mortem release of choline by the brain was measured, and the sources of this choline were, sequentially, acetylcholine, glycerophosphoryl-choline, and phospholipids.