A selective and sensitive high-performance liquid chromatographic method for the analysis of midazolam and its unconjugated 1-hydroxy metabolite in plasma samples was developed. The compounds were extracted from plasma by a liquid-liquid extraction procedure with diethyl ether. Mean analytical recoveries were 87% and 86% at a concentration of 300 ng/ml for midazolam and 1-hydroxymidazolam, respectively, and the quantification limit was 2 ng/ml for a plasma volume of 1 ml. The separation of midazolam, 1-hydroxymidazolam and flurazepam (internal standard) was achieved on a Spherisorb 5 CN column using methanol-2(r)propanol (75:25, v/v) containing 0.015% perchloric acid at a flow-rate of 1.5 ml/min. The method is sensitive enough for monitoring midazolam and also the unconjugated form of the active metabolite in plasma during pharmacokinetic studies.