Although the heart is considered a relatively pure source of m2 muscarinic receptors, the possible expression of other muscarinic receptor genes at discrete sites within the myocardium or by intrinsic cardiac ganglia had not been evaluated. Accordingly, the present study used in situ hybridization histochemistry with 35S-labeled oligonucleotide probes to address this issue. Initial experiments demonstrated that the localization of m2 mRNA was similar to that reported for muscarinic receptors labeled with the nonselective muscarinic antagonist quinuclidinyl benzilate; however, there were two important exceptions. The conducting system contained less message than expected, whereas the intrinsic cardiac ganglia contained more. The mismatch between muscarinic receptor and m2 mRNA densities in the conducting system could not be explained by the local expression of other muscarinic receptor genes, since m1, m3, and m4 mRNAs were not detected at this or any other site within the myocardium. However, the presence of a high density of prejunctional muscarinic receptors in the conducting system would be consistent with such a mismatch. Surprisingly, the intrinsic cardiac ganglia contained more than four times as much m2 mRNA as found in the atria. This level of message may be necessary for the production of prejunctional receptors on cholinergic nerve fibers within the heart and receptors localized to the ganglion cell bodies. The ganglia also contained smaller amounts of m1 and m4 mRNAs. These observations suggest that prejunctional muscarinic receptors could have a prominent role in regulating cholinergic neurotransmission in the conducting system and that multiple muscarinic receptors are present in the intrinsic cardiac ganglia.