The rate of metabolism of acetylcholine (ACh) in mouse brain in vivo has been estimated utilizing a radio gas chromatographic approach and pulse labeling with radioactive phosphorylcholine. A kinetic model describing the change with time of the plasma choline (Ch), brain Ch and ACh specific radioactivities as interdependent variables is postulated. The experimental results are analyzed kinetically according to this model using two methods: the finite difference method described by Neff et al. (J. Pharmacol. Exp. Ther. 176: 701-710, 1971) and a simple graphic method based on the transformation of the data in terms of the decline of plasma Ch radioactivity described in this paper. The accuracy of the latter is evaluated by the classic method of the least mean squares. Calculations with the two methods yield comparable results. This method is simple and can be used to study drug effects on brain ACh turnover rate. ACh turnover rate in mouse brain has been calculated to be 0.36 mumol/g/hr with the latter method, whereas the finite difference method yields slightly lower values. Because of the compartmentalization of brain Ch and the inherent assumptions, the new method can be used only for data collected during the exponential decline of plasma radioactive choline.