Novel synthetic opioids continue to emerge on recreational drug markets worldwide. In response to legislative bans on fentanyl analogues, non-fentanyl structural templates, such as 2-benzylbenzimidazoles ('nitazenes'), are being exploited to create new μ-opioid receptor (MOR) agonists. Here, we pharmacologically characterize an emerging cyclic analogue of etonitazene, called N-pyrrolidino etonitazene (etonitazepyne), using in vitro and in vivo methods. A series of analytically confirmed fatalities is described to complement preclinical findings. Radioligandbinding assays in rat brain tissue revealed that N-pyrrolidino etonitazene has high affinity for MOR (Ki = 4.09nM) over δ-opioid (Ki = 959nM) and κ-opioid (Ki = 980nM) receptors. In a MOR-β-arrestin2 activation assay, N-pyrrolidino etonitazene displayed high potency (EC50 = 0.348nM), similar to etonitazene (EC50 = 0.360nM), and largely exceeding the potencies of fentanyl (EC50 = 14.9nM) and morphine (EC50 = 290nM). When administered s.c. to male Sprague Dawley rats, N-pyrrolidino etonitazene induced opioid-like antinociceptive, cataleptic, and thermic effects. Its potency in the hot plate test (ED50 = 0.0017mg/kg) was tenfold and 2,000-fold greater than fentanyl (ED50 = 0.0209mg/kg) and morphine (ED50 = 3.940mg/kg), respectively. Twenty-one overdose fatalities associated with N-pyrrolidino etonitazene were found to contain low blood concentrations of the drug (median = 2.2ng/mL), commonly in the context of polysubstance use. N-Pyrrolidino etonitazene was reported as a cause of death in at least two cases, demonstrating toxicity in humans. We demonstrate that N-pyrrolidino etonitazene is an extremely potent MOR agonist that is likely to present high risk to users. Continued vigilance is required to identify and characterize emergent 2-benzylbenzimidazoles, and other non-fentanyl opioids, as they appear in the marketplace.