In this study we describe for the first time measurement of cholinesterase release from the brain "on-line" in the living animal, i.e. in a flow circuit incorporating a continuous supply of perfusate of a specific brain area, the substantia nigra. The basic approach has been to adapt a now well-established chemiluminescent assay. The different optimal physico-chemical conditions for various reaction stages of the assay were modified such that exogenous acetylcholinesterase, administered in pulses of short duration, could be detected above spontaneous hydrolysis of substrate in concentrations as low as 0.1 mU/ml. When exogenous enzyme was replaced by a constant delivery to the circuit of perfusate of the substantia nigra, a fluctuating signal was recorded, corresponding to spontaneous release of cholinesterase. In addition, potassium-evoked release of cholinesterase was seen to occur in a pulsatile fashion which would have been masked by the poor time resolution of any of the "off-line" methods. The potential value of this novel technique is discussed.