The 30-minute ratio of 1'-hydroxymidazolam:midazolam plasma concentrations has been used as a measure of midazolam clearance in liver transplant patients. This study determined if a single concentration of 1'-hydroxymidazolam or the ratio of 1'-hydroxymidazolam:midazolam could be used to predict midazolam clearance in healthy subjects. Plasma midazolam and 1'-hydroxymidazolam concentrations from three previous studies were used for analyses. Data obtained predose and at 5, 30, 60, 120, 240, 300, and 360 minutes following intravenous doses of midazolam in 61 adults were divided and used to derive and validate equations to predict midazolam clearance. Equations were derived using linear regression and then validated by comparing predicted to observed clearance. Only one equation was related to midazolam clearance as afunction of 1'-hydroxymidazolam, but it did not predict midazolam clearance (r = 0.29, p = 0.31). Single sampling of 1'-hydroxymidazolam or 1'-hydroxymidazolam:midazolam plasma concentrations cannot be used to predict midazolam clearance in healthy adults.