The two enantiomers of cispentacin, an antifungal antibiotic, are determined by reversed phase HPLC, after derivatization with Marfey's reagent, in 24 h urine samples collected from rats treated sc and iv with 20 mg/kg cispentacin racemate obtained by synthesis. The application range of the method is 25-250 mg/L for each enantiomer with a precision of 4.0-9.0%. Comparison with an authentic sample of natural origin (-)-cispentacin indicated that (-) enantiomer is excreted unchanged in smaller percentage than (+) enantiomer, which is almost completely eliminated as such.