Cholinergic mechanisms are known to play a key role in the regulation of breathing, but the distribution of muscarinic receptor (mAChR) subtypes has not been localized within brain stem respiratory nuclei. This study examined the hypothesis that mAChR subtypes are heterogeneously distributed across brain stem nuclei that control breathing. With the use of in vitro receptor autoradiography, the results provide the first selective labeling and quantitative mapping of M1, M2, and M3 mAChR subtypes in cat brain stem regions known to regulate breathing. Among brain stem nuclei known to contain respiratory-related neurons, the greatest amount of mAChR binding was measured in the lateral and medial parabrachial nuclei and the lateral nucleus of the solitary tract. Fewer mAChRs were localized in nuclei comprising the ventral respiratory group (nucleus ambiguous, retrofacial nucleus) and ventral medulla (retrotrapezoid nucleus and ventrolateral medulla). The data provide an essential first step for future studies aiming to specify the regulatory role of mAChR subtypes within brain stem respiratory nuclei.