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Summary1. The in vitro iontophoretic release of tritium-labelled acetylcholine and 5-hydroxytryptamine from large and small micropipettes and noradrenaline and D-lysergic acid diethylamide from small micropipettes was determined by liquid scintillation counting. 2. The release was directly proportional to the electrical charge passed in the range normally used in the iontophoretic study of these compounds. The transport numbers obtained for the large micropipettes were approximately double those with the small micropipettes. A very low transport number was found for D-lysergic acid diethylamide. 3. The spontaneous leakage was small and did not vary appreciably with time. 4. The iontophoretic release of acetylcholine in vitro agreed with the in vitro measurements. 5. The brain-stem tissue concentration of D-lysergic acid diethylamide after intravenous injection into intact and decerebrate cats was determined. IntroductionThe technique of micro-iontophoresis for applying putative synaptic transmitters, and centrally active drugs, to single neurones in the central nervous system is now well established. In almost all cases, however, it has been necessary to relate the effects on neuronal activity to the current passed and the time for which it was applied (that is to the electrical charge) rather than to the quantity of the substance released. It is therefore important to know (a) how much of the substance under study is released by a given quantity of electricity, (b) whether there is a linear relationship between the amount released and the product of current and time, and (c) whether variation in the size and shape of the electrode tip appreciably affects the quantity released, because this factor is important when comparing experimental results from different laboratories.