Epibatidine has been used in a wide dose range and was found to produce both nociceptive and antinociceptive effects. The different effects were partly explained by an action on multiple nicotinic receptor systems. The present study investigated the possibility that part of the action of intraspinally or subcutaneously administered (+/-) epibatidine, is mediated through an action on muscarinic receptors. Radioligand receptor assays were performed using homogenates of rat spinal cord and muscarinic M1-M5 receptors expressed in Sf9 cells. The intraspinal acetylcholine releasing effect of intraspinally and subcutaneously administered (+/-) epibatidine was studied with and without with atropine pretreatment. (+/-) Epibatidine has affinity for muscarinic receptors both in spinal cord tissue and expressed in Sf9 cells. The intraspinal administration of 160 microM (+/-) epibatidine produced an increase in acetylcholine release that was reduced by pretreatment with 100 microM atropine. Subcutaneous administration of 30 microg/kg (+/-) epibatidine produced an increase in intraspinal acetylcholine release that was not inhibited by 5 mg/kg subcutaneous atropine pretreatment. We conclude that (+/-) epibatidine, in microM concentrations, is a partial muscarinic receptor agonist that may interact with spinal muscarinic receptors to increase acetylcholine release. Epibatidine induced spinal acetylcholine release observed after subcutaneous administration appears not to be mediated via muscarinic receptor. The dual action on both nicotinic receptors and muscarinic receptors may explain the potent analgesic effect observed after epibatidine administration.