Administration of nicotinic agonists to the spinal cord leads to a variety of cardiovascular and behavioral responses. The discrete localization of afferent and efferent fibers presents a system in which occupation of nicotinic receptor subtypes might be correlated with specific pharmacologic responses. To this end, we examined [3H]cytisine and [3H]nicotine binding to membranes isolated from regions of the rat spinal cord. [3H]Cytisine showed saturable, noncooperative (nH congruent to 1) binding to a single-class of sites with a Kd of 0.44 +/- 0.01 nM and total saturable sites of 19.9 +/- 0.9 fmol/mg of protein. [3H]Cytisine binding to membranes from intermediolateral cell column and dorsal and ventral sections of the lumbosacral regions each revealed a single class of binding sites with virtually identical Kd values. However, the dorsal sections of the lumbar spinal cord contained a higher number of total binding sites than ventral lumbar or intermediolateral sections. The rank order potencies of the nicotinic agonists competing for [3H]cytisine binding was cytisine > I-nicotine > N-methylcarbamylcholine > dimethylphenylpiperazinium > acetylcholine > d-nicotine > carbamylcholine. Competitive antagonists also competed with high affinities (Ki as low as nanomolar) with the order of potencies being alpha-lobeline > or = dihydro-beta-erythroidine >> methyllycaconitine, whereas the channel blockers, mecamylamine and hexamethonium, only competed at concentrations > or = 100 microM. Competitive ganglionic blockers such as d-tubocurarine or trimethaphan and neurotoxins such as alpha-bungarotoxin, alpha-cobratoxin or neuronal bungarotoxin had weak affinities for cytisine sites. Similar to [3H]cytisine, [3H]nicotine also revealed a saturable single class of binding sites, but of lower affinity. The rank order of Ki values of the agonists, antagonists and neurotoxins for competing with [3H]nicotine binding was similar to the order for [3H]cytisine. Nicotinic receptors in the spinal cord membrane show a specificity for both agonists and antagonists that differ from neuronal receptors in the ganglia or the regions of the brain characterized to date. These findings, when correlated with the pharmacological responses documented in the comparison article suggests that the spinal nicotinic receptors may define a new member of the neuronal nicotinic receptor family.