We have previously localized cholinergic cell bodies and fibers within the midbrain of the chicken with choline acetyltransferase immunohistochemistry. In a continuing effort to characterize the central cholinergic system, the present study examines the distribution of various nicotinic acetylcholine receptors in the forebrain and midbrain of the chicken. The binding of 3H-nicotine, 125I-kappa-bungarotoxin, and 125I-alpha-bungarotoxin was localized by film autoradiography in adjacent sections of the adult chicken brain, allowing a comparison of the distribution of different classes of nicotinic binding sites within the brain. Although all three ligands were often co-localized, there were areas that bound 3H-nicotine but not the 125I-neurotoxins, or vice versa. Very high densities of all three ligands were found in the hyperstriatum ventrale; the nucleus geniculatus lateralis, pars ventralis; the griseum tectale; the nucleus dorsolateralis anterior thalami; the nucleus lentiformis mesencephali, pars lateralis and pars medialis; the periventricular organ; and the stratum griseum et fibrosum superficiale, layer f of the optic tectum. The nucleus spiriformis lateralis had the highest levels of 3H-nicotine binding in the chicken brain, but it did not bind either of the two snake neurotoxins. On the other hand, high levels of both 125I-alpha-bungarotoxin and 125I-kappa-bungarotoxin binding were found in the nucleus semilunaris and the nucleus ovoidalis, but these areas contained little or no 3H-nicotine binding. No unique 125I-kappa-bungarotoxin sites, unrecognized by 125I-alpha-bungarotoxin, were identified by the low resolution autoradiography performed in this study. In general, nicotinic receptors were found in areas that have been reported to contain cholinergic cell bodies or fibers. Comparison of our results with the expression of neuronal nicotinic receptor subunits, as determined by in situ hybridization, suggests that many of the high affinity 3H-nicotine sites are localized presynaptically, as, for example, in the retinorecipient nuclei and the nucleus interpeduncularis. The lack of 125I-kappa-bungarotoxin binding in the presence of alpha-bungarotoxin indicates that the chicken brain has only very low levels of a unique kappa-bungarotoxin site. This is in marked contrast to chicken, frog, and rat autonomic ganglia, where a unique kappa-neurotoxin-sensitive receptor has been identified and shown to mediate nicotinic neurotransmission.