When clonal rat pheochromocytoma PC12h cells were cultured in a hormone-supplemented serum-free medium, the carbamylcholine-elicited catecholamine release from cells cultured in serum-free medium was completely abolished. On the other hand, the high potassium-induced catecholamine release was not changed, even in PC12h cells cultured in serum-free medium. The lack of carbamylcholine sensitivity was confirmed directly by measuring carbamylcholine-induced 22Na influx, which was completely abolished in PC12h cells cultured in serum-free medium. The loss of carbamylcholine-induced 22Na influx seemed to obey nearly first-order kinetics and was fully restored upon a re-exposure to serum. The half-time for the loss was about 1 day, and the cultivation for 5 days in serum-free medium caused a 95% decrease of the nicotinic sensitivity in PC12h cells. The carbamylcholine-induced 45Ca influx into cells also was lost due to the serum-free cultivation. By contrast, the binding of alpha-bungarotoxin, which is an antagonist of nicotinic acetylcholine receptor in muscular cells, remained and did not change in PC12h cells cultured, even in the serum-free medium. In addition, veratridine-dependent 22Na influx and high potassium-induced 45Ca influx into cells, and high potassium-induced 86Rb efflux from cells cultured in the serum-free medium were also perfectly preserved. These results suggest that PC12h cells cultured in the serum-free medium seemed to be a useful model for comparing the differential mechanisms between acetylcholine sensitivity and other membranous functions on this cell.