The nicotinic agonists, nicotine and cytisine, when administered intrathecally elicit an increase in blood pressure and heart rate as well as a nociceptive response. By using a novel microdialysis procedure, in which a 4-cm dialysis tubing is inserted proximal to the intrathecal injection site, we have examined the release of excitatory amino acids associated with agonist stimulation. The nicotinic agonists, nicotine, cytisine and epibatidine, elicit dose-dependent increases in spinal release of Asp and Glu. The rank order of potencies of the nicotinic agonists in eliciting the cardiovascular and irritation responses correlates with the order of agonist potency in inducing Asp and Glu release in spinal microdialysates. In addition, a correlation is observed between the nociceptive and blood pressure response evoked by the nicotinic agonists and the spinal release of the excitatory amino acids. By examining the position of the permeable dialysis surface in relation to the agonist injection port, we found that the response to nicotine is localized to 5 to 10 mm distances, whereas the cytisine response may be elicited over longer distances. The marked desensitization observed upon repeated administration of cytisine is also reflected in diminished amino acid release. Amino acid release by nicotine can be antagonized by a channel blocking antagonist, mecamylamine, and by a competitive antagonist, dihydro-beta-erythroidine. Mecamylamine also inhibited the amino acid release elicited by epibatidine and cytisine. Hence, excitatory amino acid release is an appropriate response marker for the cellular events associated with nicotinic receptor stimulation in the spinal cord.