We have isolated a mutant m5 muscarinic receptor that mediates robust functional responses in the absence of agonists. This constitutively active receptor was isolated from a library of receptors containing randomly introduced mutations in the sixth transmembrane domain and contains the substitutions serine 465 for tyrosine and threonine 486 for proline. Although these individual residues are not conserved in other G-protein-coupled receptors, they are predicted to be at the junction between the sixth transmembrane domain and the last extracellular loop. The mutant receptor (CAm5) was subjected to detailed pharmacological analysis. All of the antagonists tested (atropine, quinuclidinyl benzilate, N-methyl scopolamine, 4-diphenylacetoxy-N-methylpiperidine and pirenzepine) fully suppressed both the constitutive and agonist-induced activities of CAm5 revealing that these ligands are negative antagonists (inverse agonists). The potency of these ligands was similar at the mutant and wild-type receptors, suggesting that the antagonist binding site of this receptor is unchanged. The mutant had increased sensitivity to the agonists carbachol, arecoline, and McN-A-343 as measured both by functional response and by radioligand binding. These effects are explained and predicted by a model in which the primary effect of the mutations is to alter a spontaneous equilibrium existing between the active and inactive states of the receptor.