The M3 muscarinic acetylcholine (ACh) receptor (M3R), a prototypic class A biogenic amine G protein-coupled receptor (GPCR), regulates various important functions of the CNS and many of the effects of ACh released from peripheral parasympathetic nerves. Agonist-activated M3Rs preferentially couple to G proteins of the Gq family but are also able to bind β-arrestins. The recruitment of β-arrestins by the activated M3R disrupts receptor/G protein coupling and promotes M3R internalization, at least in certain cell types. Of note, numerous studies have shown that GPCR-recruited β-arrestins can act as signaling molecules in their own right. These findings raise the question to which extent the recruitment of β-arrestins by activated M3Rs contributes to the physiological responses triggered by M3R activation. A better understanding of the potential physiological relevance of M3R-mediated β-arrestin recruitment may guide the development of so-called biased M3R ligands that preferentially promote either G protein signaling or β-arrestin recruitment. For this reason, the present study aimed to develop two biased mutant M3Rs with opposing functional properties. One of the mutant M3Rs selectively activates G proteins, while the other one preferentially recruits β-arrestins. Here, we report an initial functional characterization of this pair of mutant M3Rs in cultured cells. The future use of these new molecular tools for generating M3R knockin mice or for structural studies may pave the way for the clinical use of biased M3R ligands endowed with high therapeutic efficacy and a favorable side effect profile.