Incubation of minces prepared from whole brains of mice in high (25 mM) potassium (HK) medium resulted in the synthesis and release of more than 4-times the acetylcholine (ACh) formed and released from non-depolarized minces incubated in low (4 mM) potassium (LK) medium; however, potassium (K)-depolarized minces. The calcium (Ca)-dependent release of ACh evoked by K-induced depolarization appears to consist primarily of newly-formed transmitter, since this release was inhibited within 1 min after the addition of 0.1 microM hemicholinium-3 (HC-3) and was promptly reversed by choline. In contrast, the Ca-independent spontaneous release of ACh from non-depolarized minces was reduced only when the concentration of HC-3 was increased to 10 microM. The ethyl acetal derivative of the hemiacetal tautomer of HC-3 and DMAE, the tetraethyl ether of the seco tautomer of HC-3, were equipotent to HC-3 as inhibitors of ACh synthesis by non-depolarized minces but only 1/3 as active as the parent compound on K-depolarized minces. The methyl and n-propyl acetal derivatives of hemiacetal HC-3 were significantly less active than HC-3 as inhibitors of ACh synthesis by both K-depolarized and nondepolarized minces. The hemicholinium acetals and DMAE proved to be significantly more active than HC-3 as postjunctional inhibitors of ACh-induced contractions of the frog rectus abdominis muscle. Inhibitory activities of the hemicholiniums on choline acetyltransferase and cholinesterases were low and did not account for differences in prejunctional and postjunctional activities. The implications of these findings are discussed in terms of the active moiety responsible for the primary action of HC-3 on ACh synthesis.