Previous studies have shown that a single dose of methylnaltrexone, a unique peripheral opioid antagonist, reverses opioid-induced gut hypomotility in humans. Because repeated drug doses are likely to be needed to treat patients with opioid-induced or postsurgical bowel dysfunction, the authors have now examined the safety, pharmacological activity, and pharmacokinetics of a multiple-dose regimen of methylnaltrexone, administered as 12 consecutive intravenous doses (0.3 mg/kg every 6 hours) in 12 healthy subjects. Steady state was achieved rapidly, and after repeated dosing for 3 days, methylnaltrexone decreased oral-cecal transit time from a pretreatment baseline value of 101.3 +/- 29.4 min (mean +/- SD) to 82.5 +/- 20.7 min. Maximum observed plasma concentrations, measured 5 minutes postdose, were 538 +/- 237 and 675 +/- 180 ng/mL after doses 1 and 2, respectively. Based on 6-hour sampling periods, the plasma half-life, 2.5 +/- 0.5 and 2.9 +/- 0.9 hours following the 1st and 12th doses, respectively, was unchanged at steady state. There was essentially no accumulation of methylnaltrexone, based on the ratio of AUC values after doses 12 and 1. This study showed that repeated administration of intravenous methylnaltrexone is well tolerated in humans, with no significant adverse events or changes in opioid subjective ratings and no clinically noteworthy alterations in pharmacokinetics. The observation of a significant reduction in the gut transit time after repeated administration of methylnaltrexone to these opioid-naive volunteers suggests that endogenous opioids modulate human gut motility.