A new system for the study of the autonomic actions of nicotine in the rat has been developed. It is the oxotremorine-contracted longitudinal muscle of the rat terminal colon, which relaxes transiently in the presence of nicotine. This effect is mediated by nicotinic cholinergic receptors on neuronal structures since it was blocked by hexamethonium and tetrodotoxin. Other nicotinic agonists also caused the muscle strip to relax. Some of these agents 1) provided a greater maximal effect that nicotine, 2) did not show complete cross-desensitization with nicotine and 3) had tetrodotoxin-insensitive components to their action. These agents therefore do not operate solely through the same receptor nicotine does. Nicotine appears to cause relaxation by releasing a nonadrenergic inhibitory substance since 1) alpha and beta adrenergic antagonists are ineffective against nicotine and 2) nicotine retains activity on preparations from reserpinized animals. Nicotine does not cause relaxation by releasing adenosine or ATP since preparations maximally relaxed by adenosine or ATP relax further with nicotine.