1. Brain cortex slices from rats injected i.p. with urethane (1 g/kg), chloral hydrate (350 mg/kg) or physostigmine (0.75 mg/kg) were examined for acetylcholine (ACh) content, cholinesterase (total enzyme) activity and formation of (14)C-ACh from carbon (14)-uniformly labelled glucose (U-(14)C-D-glucose) in the presence of 0.01 mM physostigmine.2. Slices from rats treated with urethane, chloral hydrate, or physostigmine contained significantly higher concentrations of ACh than slices from untreated animals.3. Only slices from physostigmine-treated rats had a significantly lower cholinesterase activity.4. Slices from urethane- or chloral hydrate-treated animals formed significantly less (14)C-ACh than slices from untreated or physostigmine-treated rats when incubated in 4 mM K(+) medium. In an ACh-releasing medium (31 mM K(+)) slices from rats treated with urethane or chloral hydrate and slices from untreated rats formed similar amounts of (14)C-ACh.5. Slices from rats treated with atropine (25 mg/kg) or pentylenetetrazol (75 mg/kg) had a similar ability to form (14)C-ACh as slices from untreated animals when incubated in either 4 or 31 mM K(+) medium.6. These findings suggest that the intraneuronal ACh concentration is a limiting factor in the regulation of ACh synthesis.