The chronic pain and opioid epidemics have created an urgent need for effective, non-opioid analgesics. There is considerable evidence that the cholinergic system could be a promising target for novel drugs. Cholinesterase inhibitors and nonspecific muscarinic acetylcholine receptor (mAChR) agonists are clearly antinociceptive in humans and animal models, but adverse parasympathetic side effects limit clinical utility. The mAChR family includes five subtypes (M1-M5) with different physiologic roles. Prior studies indicate that the M2 and M4 subtypes mediate analgesia, while the M2 and M3 subtypes mediate most deleterious side effects. Therefore, an M4-selective compound might provide analgesia without prohibitive side effects. An M4 PAM, VU0467154, significantly reduced nocifensive behavior in both phases of the formalin assay in mice. However, dose-dependent motor impairment was evident in the open field and rotarod assays. A peripherally-restricted derivative of VU0467154 reduced nocifensive behavior in the second phase of the formalin assay by approximately 50% but did not cause motor impairment. These data suggest that both peripheral and central M4 mAChRs are potential targets for novel analgesics. Further studies are needed to determine the anatomic location and downstream effectors of M4 receptors involved in pain modulation. This project was supported by a grant from Foundation for Anesthesia Education and Research (to KNG)