1. The inhibition by pancuronium of acetylcholinesterase (AChE) and of plasma cholinesterase (ChE) was investigated in vitro regarding a) the sensitivity of both enzymes; b) the mechanism and constants of inhibition; and c) the relationship between the neuromuscular blocking and the anticholinesterase activity of pancuronium. 2. Pancuronium is a reversible inhibitor of both AChE and ChE. The inhibitory potency regarding ChE ([I]50=2.7 X 10(-7) M; Ki=4.2 X 10(-8) M) is highly selective and about 1000-fold higher than compared to AChE ([I]50=2.4 X 10(-4) M; Ki=3.5 X 10(-5) M). 3. The kinetic analysis by means of an Lineweaver-Burk plot and an Arunlakshana-Schild plot displayed a pure competitive mechanism of inhibition. 4. The inhibition of AChE and ChE is thought to be induced by a reversible binding of pancuronium to the anionic subsite of the active center, thus decreasing the formation of the primary enzyme-substrate complex. 5. The clinical administration of pancuronium for muscular relaxation during anaesthesia (0.01-0.08 mg/kg) will result in a concentration of approximately 10(-7)=10(-6) M in the extracellular fluid. Thus, an inhibition of plasma ChE can be expected to occur under clinical conditions, however, probably without practical significance.