Slices of rat caudate nuclei were incubated in saline media containing choline, paraoxon, unlabelled glucose, and [1,5‐¹⁴C]citrate, [1‐¹⁴C‐acetyl]carnitine, [1‐¹⁴C]acetate, [2‐¹⁴C]pyruvate, or [U‐¹⁴C]glucose. The synthesis of acetyl‐labelled acetylcholine (ACh) was compared with the total synthesis of ACh. When related to the utilization of unlabelled glucose (responsible for the formation of unlabelled ACh), the utilization of labelled substrates for the synthesis of the acetyl moiety of ACh was found to decrease in the following order: [2‐¹⁴C]pyruvate > [U‐¹⁴C]glucose > [1‐¹⁴C‐acetyl]carnitine > [1,5‐¹⁴C]citrate > [1‐¹⁴C]acetate. The utilization of [1,5‐¹⁴C]citrate and [1‐¹⁴C]acetate for the synthesis of [¹⁴C]ACh was low, although it was apparent from the formation of and ¹⁴C‐labelled lipid that the substrates entered the cells and were metabolized. The utilization of [1,5‐¹⁴C]citrate for the synthesis of [¹⁴C]ACh was higher when the incubation was performed in a medium without calcium (with EGTA); that of glucose did not change, whereas the utilization of other substrates for the synthesis of ACh decreased. The results indicate that earlier (indirect) evidence led to an underestimation of acetylcar‐nitine as a potential source of acetyl groups for the synthesis of ACh in mammalian brain; they do not support (but do not disprove) the view that citrate is the main carrier of acetyl groups from the intramitochondrial acetyl‐CoA to the extramitochondrial space in cerebral cholinergic neurons.