This study was conducted to determine the pharmacokinetics and pharmacodynamics of pyridostigmine given as 30 mg of pyridostigmine bromide every 8 hours in healthy subjects. Plasma pyridostigmine concentration and red blood cell acetylcholinesterase activity were measured in blood samples collected during a 3-week period. Population analysis was performed using standard pharmacokinetic and pharmacodynamic models with the nonlinear mixed-effect modeling software (NONMEM). The pharmacokinetic model that best fit the pyridostigmine plasma levels was a two-compartment open model with first-order absorption, a lag time, and first-order elimination from the central compartment. The pharmacodynamic model that best fit red blood cell acetylcholinesterase activity was an inhibitory Emax model with an effect compartment linked to the central compartment. The results showed that the pharmacokinetics of pyridostigmine bromide are both gender and weight dependent. The pharmacodynamic effect does not lag significantly from the plasma concentration and returns to near normal within 8 hours. With the present dosage regimen of 30 mg every 8 hours, 30% of individuals may not have red blood cell acetylcholinesterase inhibition > 10% at the time of the trough.