We report on the determination of choline and acetylcholine via biocentrivoltammetry. This method combines centrifugation and voltammetry and is based on a carbon paste electrode modified with acetylcholinesterase and choline oxidase. The electrode was placed at the bottom of a biocentrivoltammetric cell. Acetylcholine and choline are accumulated on the enzyme electrode via centrifugative forces, upon which a direct voltammetric scan is applied. Reaction time, pH values, quantities of enzyme and centrifugation parameters were optimized. A linear response is obtained in the 0.07 to 10 mu M concentration range of acetylcholine, and a limit of detection as low as 0.5 mu M. The linear range is between 0.1 and 500 mu M for choline. The method was applied to the determination of acetylcholine and choline in spiked serum samples.