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1. Acetylcholine (ACh), cholinesterases and choline acetyltransferase (choline acetylase) were estimated in the neural lobe and hypothalamus of the adult male rabbit. Acetylcholine was also estimated in the neural lobes and hypothalami of some other mammals.

2. Acetylcholine-like activity was measured by bio-assay using the leech dorsal muscle preparation.

3. Characterization experiments indicated that about 90% of the activity measured was due to acetylcholine.

4. Mean acetylcholine content in the neural lobe of the rabbit, after extraction with perchloric acid, was 4·38 ± 0·98 µg/g fresh tissue, and 4·87 ± 1·53 µg/g in the hypothalamus.

5. Acetylcholine was also found to be present, in comparable concentrations, in the neural lobe of man and in the neural lobes and hypothalami of ox, rat and hedgehog.

6. Acetylcholinesterase, present in the neural lobe and hypothalamus of the rabbit, hydrolysed 1·74 ± 0·11 µ-moles of substrate/min/g and 3·78 ± 0·60 µ-moles substrate/min/g fresh tissue respectively.

7. The concentration of butyrylcholinesterase was about one tenth that of acetylcholinesterase in both tissues.

8. Choline acetyltransferase present in the neural lobe and in the hypothalamus synthesized 87 ± 22 µg ACh/hr/g fresh tissue and 378 ± 149 µg respectively.