Digoxigenin-labeled RNA probes and in situ hybridization histochemistry were used to examine choline acetyltransferase gene expression in the rat central nervous system. Hybridization signal was present only in brain sections processed with the antisense riboprobe. The sense probe did not yield labeling, further validating the specificity of tissue reactivity. Telencephalic neurons containing the mRNA for the cholinergic synthetic enzyme were found in the caudate-putamen nucleus, nucleus accumbens, olfactory tubercule, islands of Calleja complex, medial septal nucleus, vertical and horizontal limbs of the diagonal band, substantia innominata, nucleus basalis, and nucleus of the ansa lenticularis. Some somata evincing hybridization signal were observed in the anterior amygdalar area, and an occasional such cell was seen in the basolateral and central amygdalar nuclei. Neurons in the cerebral cortex, hippocampus, and primary olfactory structures did not demonstrate hybridocytochemically detectable amounts of choline acetyltransferase mRNA. Thalamic cells were devoid of reactivity, with the exception of several neurons located primarily in the ventral two-thirds of the medial habenula. A few somata labeled with riboprobe were found in the lateral hypothalamus, caudal extension of the internal capsule, and zona incerta. Neurons in the pedunculopontine and laterodorsal tegmental nuclei were moderately reactive, whereas cells of the parabigeminal nucleus exhibited a very weak hybridization signal. No somata in the brainstem raphe nuclei, including raphe obscurus and raphe magnus, were observed to bind riboprobe. In contrast, motor neurons of the cranial nerve nuclei demonstrated relatively large amounts of choline acetyltransferase mRNA. Putative cholinergic somata in the ventral horns and intermediolateral cell columns of the spinal cord were also labeled with riboprobe, as were a few cells around the central canal. We conclude that hybridocytochemistry with digoxigenin-labeled riboprobes confirms the existence of cholinergic neurons (i.e. those that synthesize and use acetylcholine as a neurotransmitter) in most of the neural regions deduced to contain them on the basis of previous histochemical and immunocytochemical data. Notable exceptions are the cerebral cortex and hippocampus, which do not possess neurons expressing detectable levels of choline acetyltransferase mRNA.