Objectives A volunteer trial was performed to compare the pharmacokinetics of 5 drugs—warfarin, ZK253 (Schering), diazepam, midazolam, and erythromycin—when administered at a microdose or pharmacologic dose. Each compound was chosen to represent a situation in which prediction of pharmacokinetics from either animal or in vitro studies (or both) was or is likely to be problematic. Methods In a crossover design volunteers received (1) 1 of the 5 compounds as a microdose labeled with radioactive carbon (carbon 14) (100 μg), (2) the corresponding 14C‐labeled therapeutic dose on a separate occasion, and (3) simultaneous administration of an intravenous 14C‐labeled microdose and an oral therapeutic dose for ZK253, midazolam, and erythromycin. Analysis of 14C‐labeled drugs in plasma was done by use of HPLC followed by accelerator mass spectrometry. Liquid chromatography‐tandem mass spectrometry was used to measure plasma concentrations of ZK253, midazolam, and erythromycin at therapeutic concentrations, whereas HPLC‐accelerator mass spectrometry was used to measure warfarin and diazepam concentrations. Results Good concordance between microdose and therapeutic dose pharmacokinetics was observed for diazepam (half‐life [t½] of 45.1 hours, clearance [CL] of 1.38 L/h, and volume of distribution [V] of 90.1 L for 100 μg and t½ of 35.7 hours, CL of 1.3 L/h, and V of 123 L for 10 mg), midazolam (t½ of 4.87 hours, CL of 21.2 L/h, V of 145 L, and oral bioavailability [F] of 0.23 for 100 μg and t½ of 3.31 hours, CL of 20.4 L/h, V of 75 L, and F of 0.22 for 7.5 mg), and development compound ZK253 (F = Clinical Pharmacology & Therapeutics (2006) 80, 203–215; doi: 10.1016/j.clpt.2006.05.008