A direct chiral HPLC method has been developed and validated to quantitate the enantiomers of MDL 74 156, the primary metabolite of dolasetron mesylate, in human plasma over the concentration range 1.70–340 ng ml −1. Dolasetron mesylate is a 5-HT 3 receptor antagonist that is currently being developed as an antiemetic. Both enantiomers of MDL 74 156 and the internal standard (granisetron) were first extracted from alkanized plasma using methyl t-butyl ether. The analytes were then back-extracted into formic acid, separated on a ovomucoid-bonded HPLC column, and detected by native fluorescence (excitation wavelength of 274 nm and emission wavelength of 345 nm). The completed validation demonstrated the method to be accurate, precise, and specific for the direct quantitation of MDL 74 156 enantiomers in human plasma. This procedure has been used on a routine basis to quantify the relative concentrations of each enantiomer of MDL 74 156 in both oral and intravenous pharmacokinetic studies of dolasetron mesylate in normal human volunteers.