An effective and selective procedure for the extraction of ambenonium chloride (AMBC) from serum using a weak cation-exchange extraction cartridge has been developed. The solid-phase extraction procedure permitted the extraction of AMBC from serum without adhesion to materials such as the containers. A 200-microliter volume of the eluate could be directly injected on to a reversed-phase ion-pair high-performance liquid chromatographic column. The recovery was in the range 97-100%. The limit of detection for AMBC was 0.5 ng/ml in serum (signal-to-noise ratio = 3). The method was used to determine the serum concentration of AMBC in patients with myasthenia gravis. The method would be useful for monitoring AMBC in serum in order to study its pharmacokinetic behaviour in patients under oral administration therapy.