The goal of the present study was to identify the cells containing mRNA coding for the m2 subtype of muscarinic cholinergic receptors in the rat brain. In situ hybridization histochemistry was used, with oligonucleotides as hybridization probes. The distribution of cholinergic cells was examined in consecutive sections with probes complementary to choline acetyltransferase mRNA. Furthermore, the microscopic distribution of muscarinic cholinergic binding sites was examined with a non-selective ligand ([3H]N-methylscopolamine) and with ligands proposed to be M1-selective ([3H]pirenzepine) or M2-selective ([3H]oxotremorine-M). The majority of choline acetyltransferase mRNA-rich (i.e. cholinergic) cell groups (medial septum-diagonal band complex, nucleus basalis, pedunculopontine and laterodorsal tegmental nuclei, nucleus parabigeminalis, several motor nuclei of the brainstem, motoneurons of the spinal cord), also contained m2 mRNA, strongly suggesting that at least a fraction of these receptors may be presynaptic autoreceptors. A few groups of cholinergic cells were an exception to this fact: the medial habenula and some cranial nerve nuclei (principal oculomotor, trochlear, abducens, dorsal motor nucleus of the vagus). Furthermore, m2 mRNA was not restricted to cholinergic cells but was also present in many other cells throughout the rat brain. The distribution of m2 mRNA was in good, although not complete, agreement with that of binding sites for the M2 preferential agonist [3H]oxotremorine-M, but not with [3H]pirenzepine binding sites. Regions where the presence of [3H]oxotremorine-M binding sites was not correlated with that of m2 mRNA are the caudate-putamen, nucleus accumbens, olfactory tubercle and islands of Calleja. The present results strongly suggest that the M2 receptor is expressed by a majority of cholinergic cells, where it probably plays a role as autoreceptor. However, many non-cholinergic neurons also express this receptor, which would be, presumably, postsynaptically located. Finally, comparison between the distribution of m2 mRNA and that of the proposed M2-selective ligand [3H]oxotremorine-M indicates that this ligand, in addition to M2 receptors, may also recognize in certain brain areas other muscarinic receptor populations, particularly M4.