The effects of (-)-hydroxycitrate (OHC) and citrate on the concentration of acetylcoenzyme A (acetyl-CoA) and acetylcholine (ACh) in the tissue and on the release of ACh into the medium were investigated in experiments on slices of rat caudate nuclei incubated in media with 6.2 or 31.2 mM K+, 0 or 2.5 mM Ca2+, and 0, 1, or 10 mM EGTA. OHC diminished the concentration of acetyl-CoA in the slices under all conditions used; in experiments with 2.5 mM OHC, the concentration of acetyl-CoA was lowered by 25-38%. Citrate, in contrast, had no effect on the level of acetyl-CoA in the tissue. Although both OHC and citrate lowered the concentration of ACh in the slices during incubations with 6.2 mM K+ and 1 mM EGTA, they had different effects on the content of ACh during incubations in the presence of Ca2+. The concentration of ACh in the slices was increased by citrate during incubations with 2.5 mM Ca2+ and 31.2 or 6.2 mM K+, but it was lowered or unchanged by OHC under the same conditions. The release of ACh into the medium was lowered or unchanged by OHC and lowered, unchanged, or increased by citrate. It is concluded that most effects of OHC on the metabolism of ACh can be explained by the inhibition of ATP-citrate lyase; with glucose as the main metabolic substrate, ATP-citrate lyase appears to provide about one-third of the acetyl-CoA used for the synthesis of ACh. Experiments with citrate indicate that an increased supply of citrate may increase the synthesis of ACh. The inhibitory effect of citrate on the synthesis of ACh, observed during incubations without Ca2+, is interpreted to be a consequence of the chelation of intracellular Ca2+; this interpretation is supported by the observation of a similar effect caused by 10 mM EGTA.