1. The synthesis of carbon-14 labelled acetylcholine ((14)C-ACh) from carbon-14 uniformly labelled glucose (U.L. (14)C-glucose) under different conditions has been studied.2. Increasing KCl concentration from 4 mM to 31 mM was associated with a large increase in the amount of (14)C-ACh in the medium and a significant decrease in the amount of (14)C-ACh in the tissue. This effect was observed in slices and minces but not in homogenate.3. Increasing KCl concentration from 4 mM to 31 mM resulted in the release of pre-formed (14)C-ACh.4. Elevated pre-incubation levels of ACh in slices incubated in 4 mM KCl medium was associated with a decrease in the formation of (14)C-ACh.5. These findings are consistent with the hypothesis that the concentration of ACh in the vicinity of the site of synthesis regulates the rate of ACh formation.