This study presents, to our knowledge, the first on-line measurement of acetylcholinesterase (AcChoEase) release from brain tissue. It is now well established that a soluble form of the enzyme is released from central nervous system neurons, and it has been proposed on indirect grounds that such release may occur not only from presynaptic terminals but also from the dendrites of dopamine-containing nigrostriatal neurons. We have used a chemiluminescent reaction to examine the real-time release of AcChoEase from the substantia nigra in vitro in brainstem slices. The light emission was captured by two fiber optic systems, one in direct contact with the brain slice from below and the second 4-mm above the slice, allowing simultaneous imaging of the emitted light and quantitative photometry. It was determined that the light signals are not due to the spontaneous hydrolysis of acetylcholine or the presence of free choline, but are caused by the enzymatic action of AcChoEase. Using this technique, it can be directly shown that AcChoEase is spontaneously released from the soma or dendrites of nigral neurons. The release of the enzyme, which is stored in the subcisternal dendritic compartment, is resistant to blockade of voltage-dependent sodium conductances, is calcium dependent, and can be increased by addition of potassium to the bathing solution. The procedure we describe here will make it possible to study the release of endogenous AcChoEase on a time-scale close to that over which it functions.