This work reports two methods developed for the separation and determination of the enantiomers of the new alkaloid malacitanine (MLC) and the determination of the enantiomeric purity in mixtures. First, the isomers were separated using a Chirex 3020 (250 mm x 4.6 mm, 5 mu m) chiral column with a mobile phase of cyclohexane1,2-dichloroethaneethanoltrifluoroacetic acid (64:30:6:0.6, v/v/v/v) at a flow rate of 1 mL/min and fluorimetric detection. Obtained retention times were 12.4 and 15.9 min (+ and -) with a resolution Rs of 1.13. Relative standard deviations (RSDs) were 2.5 and 2.4% at the 0.5-mu g level (four determinations). Second, a nonenantioselective procedure for the determination of enantiomeric purity of MLC using a Lichrospher (R) Si-60 (250 mm x 5 mm, 5 mu m) normal phase with a mobile phase of 100% ethanol at a flow rate of 0.9 mL/min coupled to two detectors in series, fluorimetric and polarimetric. RSD of 3.3% was obtained. Calculated enantiomeric purity by chiral chromatography gave 48.6% (-)-MLC in the near racemic product. Using polarimetric signal of the nonseparated enantiomers and comparing the slopes of the calibration curves (enantiomers) from the racemic product gave 47.8% (-)-MLC content. A study of accuracy of (-)-MLC gave recoveries from 98.3 to 100.7%.