In cultured neurons dissociated from the spinal cord of fetal mouse, high concentrations of KCl (47 mM) increased choline acetyltransferase (CAT) activity up to 5.5-fold but suppressed acetylcholinesterase (AChE) activity to less than half the level of control cells. Veratridine (3 microM) also increased CAT activity 1.6-fold and suppressed AChE activity to the same level as that induced by high KCl. The increase of CAT activity by the depolarizing agents was blocked by Ca2+ antagonists (verapamil and high Mg2+) and in a low Ca2+ medium, whereas the suppression of AChE activity by high KCl was restored by the same procedures. The synthesis of radiolabeled acetylcholine from [14C]choline was also enhanced 4-fold by incubating cells in high KCl medium. Although the uptake of L-[3H]leucine and [14C]choline into the cells was slightly enhanced by high KCl medium, neither the total amount of protein nor the incorporation of L-[3H]leucine into protein was increased by high KCl medium. These observations indicate that depolarization increased CAT activity in a specific manner, that the activities of CAT and AChE changed inversely under several conditions, and that the effect of depolarization presumably was mediated by the entry of Ca2+ into neuronal cells. The findings raise the possibility that trans-synaptic input could play a crucial role in the development of the activity of cholinergic neurons in spinal cord.