Epibatidine has been shown to be the most potent nicotinic agonist in several neuronal nicotinic receptor preparations. Similar to other nicotinic agonists, intrathecal (-)-epibatidine elicits dose-dependent increases in pressor, heart rate and pain responses in rats, as well as an increase in latency to withdraw from a noxious thermal stimulus. The latter response requires higher doses and is of shorter duration, suggesting interaction with multiple subtypes of spinal nicotinic receptors. In the present study, we relate the binding properties of (+/-)-[3H]epibatidine in spinal cord membrane preparations to the cardiovascular and behavioral responses. Unlike (-)-[3H]cytisine or (-)-[3H]nicotine, (+/-)-[3H]epibatidine reveals two sites; the ratio of high affinity to low affinity sites is 2:1. The rank ordering of potencies of the nicotinic agonists in displacing (+/-)-[3H]epibatidine binding from spinal cord membranes correlates with the potencies in eliciting cardiovascular and behavioral responses upon spinal administration. The nicotinic receptor antagonists, alpha-lobeline, dihydro-beta-erythroidine and methyllycaconitine, also displayed similar rank ordering of potencies in displacing (+/-)-[3H]epibatidine, (-)-[3H]cytisine or (-)-[3H]nicotine binding to spinal nicotinic receptors. Virtually all the nicotinic analogs exhibited a Hill coefficient of less than one in competing with (+/-)-[3H]epibatidine to spinal cord membranes indicating their interaction with at least two classes of binding sites. Intrathecal (-)-epibatidine, in addition to eliciting an initial and subsequently a sustained pressor and tachycardic response, also exhibited a transient intervening bradycardia which coincided temporally with the duration of the analgesia. Repeated administration of (-)-epibatidine desensitized its responses as well as the cardiovascular and behavioral responses to spinal nicotine and cytisine. Intrathecal alpha-lobeline showed selectivity for blocking the analgesic response, whereas methyllycaconitine exhibited selectivity for the pressor and irritation responses. The NMDA receptor antagonist, AP-5, inhibited the pressor, tachycardic and irritation responses elicited by intrathecal (-)-epibatidine, confirming a role for spinal excitatory amino acids released by the nicotinic agonist.