A new HPLC method for the separation and quantification of cloprostenol enantiomers was developed. The optimized separation system consisted of Chiralcel OD-RH column and acetonitrile-sodium dihydrogenphosphate (pH 3.0; 20mM) (33:67, v/v) as the mobile phase. Baseline resolution of (+/-)-cloprostenol (R=2.16) was achieved and the analysis time did not exceed 10 min. Limits of detection and quantification were units of micromol/l at 274 nm. The respective values decreased an order of magnitude at 210 nm. The R.S.D. values obtained for the retention factor, peak area and peak height of each enantiomer were less than 2%. Conditions for semipreparative separation of the enantiomers can be achieved easily just by a small adaptation of the mobile phase composition.