Chronic nicotine administration to rats produces an increase in neuronal nicotinic receptors in the CNS. Moreover, the up-regulated sites labeled by [3H]cytisine in cerebral cortex appear to be composed exclusively of alpha4 and beta2 subunits. It is unknown whether receptor subtypes that do not bind [3H]cytisine with high affinity are also affected. In the present studies, we tested the hypothesis that nicotine treatment differentially alters the density of neuronal nicotinic receptor subtypes in rat nervous tissues. Thus, we compared the binding of [3H]cytisine with that of [3H]epibatidine to nicotinic receptors in brain, spinal cord, and adrenal gland from rats that had been injected twice daily with nicotine or saline vehicle for 10 days. Chronic nicotine treatment led to an increase in nicotinic receptor binding sites in the cerebral cortex and in the dorsal lumbar spinal cord, but not in the thalamus. It is important that virtually all of the observed increases could be accounted for by a selective effect on the fraction of receptors exhibiting high affinity for both [3H]cytisine and [3H]epibatidine. In contrast, no change in [3H]epibatidine binding was seen in the adrenal gland, a tissue that does not exhibit high-affinity [3H]cytisine binding. These data indicate that, under the conditions used here, nicotine up-regulates the alpha4beta2 nicotinic receptor subtype, which can be labeled by [3H]cystisine and [3H]epibatidine, but not non-alpha4beta2 subtypes, which can be labeled by [3H]epibatidine.