Choline acetyltransferase, a specific marker for cholinergic neurons, has been immunohistochemically localized in the mesencephalon and in the caudal diencephalon of the chicken. A complete series of transverse sections through the mesencephalon is presented. In the diencephalon, cholinergic fibers were found in the stria medullaris, the fasciculus retroflexus, and the ventral portion of the supraoptic decussation. The nucleus triangularis and the nucleus geniculatus lateralis, pars ventralis also contained cholinergic fibers. Small cholinergic cell bodies were found in the medial habenula. In the pretectum, cholinergic fibers innervated the nucleus lentiformis mesencephali and the tectal gray. The nucleus spiriformis lateralis also contained cholinergic fibers, while most of the cell bodies in the nucleus spiriformis medialis were cholinergic. In the mesencephalon, labelled fibers were found in the nucleus intercollicularis and in all layers of the optic tectum except the stratum opticum. The highest density of tectal cholinergic fibers was in the stratum griseum et fibrosum superficiale (SGFS), layer f. Radial cells located in SGFS, layer i were also cholinergic. In the isthmic nuclei, cholinergic fibers were found in the pars magnocellularis, while the pars parvicellularis and the nucleus semilunaris contained labelled cells. The oculomotor, Edinger-Westphal, trochlear, and trigeminal motor nuclei all had cholinergic cell bodies. Cholinergic axons were present in the oculomotor and trochlear nerves. In the tegmentum, cell bodies were labelled in the nucleus mesencephalicus profundus, pars ventralis, while the nucleus interpeduncularis had dense cholinergic innervation. Our localization of cholinergic cell bodies and fibers has been compared with earlier autoradiographic and anatomical studies to help define cholinergic systems in the avian brain. For example, the results indicate that the chicken may have a cholinergic habenulointerpeduncular system similar to that reported in the rat. Establishing the cholinergic systems within the avian midbrain is important for designing future neurophysiological and pharmacological studies of cholinergic transmission in this region.