SummaryThis study investigated the effect of muscarinic M1and M2receptor antagonists on the rocuronium‐induced train of four (TOF) fade and tetanic fade, respectively. Ex‐vivo phrenic nerves and diaphragms were obtained from adult Sprague‐Dawley rats and stabilized in Krebs buffer; the nerve‐stimulated muscleTOFfade was observed at 20 s intervals. For theTOFstudy, phrenic nerves and diaphragms were incubated with pirenzepine (an M1blocker) at concentrations of 0 nmol L−1(control), 10 nmol L−1(PZP10), or 100 nmol L−1(PZP100). Rocuronium was then administered incrementally until the first twitch tension had depressed by >95% duringTOFstimulation. The meanTOFratios were compared when the first twitch tensions were depressed by 40%‐50%. For the tetanic fade study, 50 Hz/5 s tetani was applied initially, 30 min after the administration of a loading dose of rocuronium and methoctramine (an M2receptor blocker, loaded at 0 μmol L−1[control], 1 μmol L−1[MET1], or 10 μmol L−1[MET10]). TheEC95of rocuronium was significantly lower in thePZP10 group than in the control group. In thePZP10 group, theTOFratios at 50% and first twitch tension depression were significantly lower than those in the control group (P=.02). During tetanic stimulation, the tetanic fade was significantly enhanced in theMET10 group compared to the other groups. This study shows that antagonists of muscarinic M1and M2receptors affect the rocuronium‐induced neuromuscular block as demonstrated by the reducedEC95andTOFratios (M1antagonist, pirenzepine) or the enhanced 50‐Hz tetanic fade (M2antagonist, methoctramine).