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1. The acetylcholine (ACh) store of cat's superior cervical ganglia was replaced with radioactive ACh by perfusion, during stimulation, with [³H]choline—Locke solution. Perfusion was continued with Locke containing unlabelled choline (Ch) (in physiological concentration) and the release of labelled and unlabelled ACh was measured.

2. Electrical stimulation of the preganglionic sympathetic nerve (20/sec or 5/sec), or stimulation by perfusing with raised K, released ACh that had a lower specific radioactivity than ganglionic ACh. The proportion of released ACh that was labelled was slightly higher when stimulation was at lower frequency or by K.

3. Preganglionic nerve stimulation released, in the first few minutes, ACh that had a specific activity 70-80% of ganglionic ACh, but after 5 min the proportion of label in the released ACh fell to 35-45% of that in the ganglion.

4. It is concluded that newly synthesized ACh is released before equilibration with preformed stores, and the significance of this to the mechanism of transmitter release is discussed.

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