We have used in situ hybridization to study the localization of muscarinic receptor subtype mRNAs and compared this with the distribution of muscarinic receptor subtypes using receptor autoradiography in tissue sections of the rabbit lung. To localize muscarinic receptor subtype mRNAs, subtype-specific synthetic oligonucleotide probes or riboprobes were generated. Two muscarinic receptor mRNAs (m3 and m4) were detected. The m3 mRNA was expressed in high concentration in tracheal smooth muscle, epithelium and blood vessels and to a lesser extent in bronchiolar smooth muscle while the m4 mRNA was abundantly expressed in bronchiolar smooth muscle and alveolar walls. No expression of m1, m2 or m5 mRNAs was found in any lung structures. This was confirmed by Northern blot analysis which revealed only a single transcript of 3.6 kb for m4 mRNA in rabbit lung. With the exception of the airway epithelium and blood vessels, there was a correspondence between mRNA localization and the distribution of muscarinic receptor subtypes visualized by [3H](-)-quinuclidinyl benzilate ([3H]QNB) autoradiographically in the presence of relatively selective muscarinic subtype antagonists (M1-M3) and direct labelling with selective [3H]AF-DX 384. The significance and the reasons for the presence of m4 mRNA and M4 receptors in the rabbit lung are uncertain and warrant further investigation.