Experiments with cats indicate that HC-3 (No. 3 of Schueler''s list of hemicholiniums, Schueler, F. W., Jour. Pharmacol. 115:127, 1955.) has no effect on the mechanisms involved in the release of acetylcholine, but blocks transmission in active cholinergic pathways by allowing the presynaptic stores of transmitter to be depleted. The toxic action of this drug may be due to this type of effect, even though the transmission of respiratory activity within the central nervous system requires the participation of cholinergic neurons. The drug appears to be only a weak inhibitor of choline acetylase, as shown by its failure to prevent the synthesis of acetylcholine by extracts of acetone powders of brain incubated with choline and acetyl-coenzyme A. Other results indicate that HC-3 strongly inhibits the transfer of choline across the cells of the avian renal tubule, a process which apparently does not involve the acetylation of choline. It is concluded that HC-3 probably interferes with acetylcholine synthesis in nervous tissue by competitively inhibiting the transport of choline to its intracellular site of acetylation.