PC12, a clonal line of rat pheochromocytoma, synthesizes, stores, and secretes dopamine and acetylcholine. The cells take up choline by a saturable process and rapidly convert the accumulated choline to acetylcholine. This choline transport has a Km of 12 microM, is Na+ and energy independent, and is relatively insensitive to hemicholinium-3 (IC50 approximately 50 microM). Different ionic conditions can modulate the choline transport. Uptake was increased by pretreatment with 55 mM K+ whereas it was decreased in the presence of 55 mM K+. Choline uptake had similar characteristics in PC12 cells that had been induced to extend neurites by treatment with nerve growth factor. In undifferentiated PC12 cells, storage of newly synthesized acetylcholine was found in bound and free compartments as evidenced from subcellular fractionation. The free pool had a faster turnover rate. Most of the newly synthesized acetylcholine was rapidly degraded in the absence of a cholinesterase inhibitor while continuous incubation with labeled choline resulted in a slow incorporation of newly labeled acetylcholine into a bound pool. The accumulation of acetylcholine in the bound pool, but not acetylcholine synthesis, was inhibited by each of several agents that are known to interfere with the generation or maintenance of proton electrochemical gradients. The newly synthesized acetylcholine could be released from PC12 cells by incubation of the cells with 55 mM K+. These properties indicate that PC12 cells are a good system for studying acetylcholine metabolism by secretory cells.