Relationship between in vivo down-regulation of cardiac muscarinic receptors and changes in their encoding mRNA was investigated. Rats were treated either once or for ten days with an irreversible inhibitor of acetylcholinesterase, followed by measurements of cardiac acetylcholinesterase, the density and affinity of muscarinic receptors, and the concentration of mRNA coding for these receptors. mRNA was quantitated using the sensitive method of DNA-excess solution hybridization. Our data indicate that while short-term treatment resulted in a marked decrease in the density of cardiac muscarinic receptors by 34%, there was no accompanying significant change in the concentration of their mRNA. In contrast, long-term inhibition of acetylcholinesterase significantly decreased the concentration of both receptors and mRNA by 40% and 29%, respectively. These results are indicative of multiple mechanisms of down-regulation of cardiac muscarinic receptors, some of which might involve alterations at the transcriptional level.