This study investigated the effect of muscarinic M1 and M2 receptor antagonists on the rocuronium‐induced train of four (TOF) fade and tetanic fade, respectively. Ex‐vivo phrenic nerves/diaphragms were obtained from adult Sprague‐Dawley rats and, stabilized in Krebs buffer, and the nerve‐stimulated muscle TOF fade was observed at 20 s intervals. For the TOF study, phrenic nerve/diaphragms were incubated with pirenzepine (an M1 blocker) at concentrations of 0 nM (control), 10 nM (PZP10), or 100 nM (PZP100). Rocuronium was then administered incrementally until the first twitch tension (T1) had depressed by >95% during TOF stimulation. The mean TOF ratios were compared when the T1 tensions were depressed by 40% to 50%. For the tetanic fade study, 50 Hz/5 s tetani were applied initial, 30 min after administering a loading dose of rocuronium, and 30 min after administering methoctramine (an M2 receptor blocker, loaded at 0 μM (control), 1 μM (MET1), or 10 μM (MET10)). The EC95 of rocuronium was significantly lower in the PZP10 than in the control group. In the PZP10 group, the TOF ratios (TOFRs) at 50s%, T1 depression were significantly lower than those in the control group (p = 0.02). During tetanic stimulation, the tetanic fade was significantly enhanced in the MET10 group compared to the other groups. The study shows that antagonists of muscarinic M1 and M2 receptors affect the rocuronium‐induced neuromuscular block evidenced as shown by the lowering of EC95 and TOFR (M1 antagonist, pirenzepine) or enhancing 50 Hz tetanic fade (M2 antagonist, methoctramine). This article is protected by copyright. All rights reserved.