A direct stereoselective reversed-phase HPLC method is described for the determination of the enantiomers of a proton pump inhibitor, pantoprazole (PAN), in human serum. The enantiomers were separated with high resolution on a cellulose-based chiral stationary phase (Chiralcel OJ-R) following on-line solid phase sample cleanup with a column-switching device. A mixture of acetonitrile and 50 mM sodium perchlorate was used as the mobile phase at a flow rate of 0.5 mliter/min. Pantoprazole enantiomers were detected by monitoring the column effluent with UV light at a wavelength of 290 nm. The calibration curve for each enantiomer was linear from 0.1 to 5.0 micrograms/mliter. Under these conditions, the determination of pantoprazole enantiomers in human serum can be achieved with satisfactory selectivity, sensitivity, precision, and accuracy. The described procedure is very simple and rapid since labor-intensive sample preparation is not required. The method was applied to the analyses of the serum samples obtained from a volunteer who received an 80-mg oral dose of racemic PAN. The samples showed the (+)/(-) isomer ratios ranging from 0.74 to 1.03 up to 6 h after dosing, indicating that there is only a small difference in the concentrations of (+)- and (-)-PAN.