The membrane of bullfrog sympathetic ganglion cells was hyperpolarized by a direct action of ACh (more than 0.5 mM) in a solution containing both nicotine (0.24 mM) and atropine (0.14 mM). This ACh hyperpolarization could be imitated by neither carbachol nor bethanechol, suggesting that the ACh hyperpolarization was the response which was produced by a specific action of ACh, which appeared to be neither nicotinic nor muscarinic. The size of ACh hyperpolarization was increased during a conditioning hyperpolarization. The ACh hyperpolarization was completely blocked by ouabain (2 times 10(-3) mM) and eliminated in the Na-free lithium solution. These aspects of the ACh hyperpolarization suggested that generation of this hyperpolarization was associated with the sodium pump. The ACh hyperpolarization seemed to be partially responsible for the production of the slow IPSP, since a part of the slow IPSP remained occasionally in the presence of both nicotine and atropine.