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A sensitive, enantioselective high-performance liquid chromatographic method has been developed for the separation and individual quantitative determination of (+)- and (-)-tramadol and (+)- and (-)-O-desmethyltramadol (M1) in plasma and urine. Extraction from plasma and urine was performed by solid-phase extraction (SPE) on disposable butyl silica (100 mg) extraction cartridges. Separation of the enantiomers of tramadol and M1 was achieved on a Chiralpak AD column containing amylose tris-(3,5-dimethylphenylcarbamate) as chiral selector. The mobile phase was isohexane-ethanol-diethylamine, 97:2.8:0.1 (v/v). Quinidine was used as internal standard. The analytes were detected by use of fluorescence detection. The limit of quantification for tramadol and M1 was 5 nM in plasma and 25 nM in urine. Recoveries were approximately 90% for tramadol and M1 in both plasma and urine. Linearity was observed for both enantiomers of tramadol and M1 in both plasma (r2 > 0.999) and urine (r2 > 0.997). The intra and inter-day precision (CV) did not exceed 6.0%. The applicability of the method was demonstrated by means of two clinical studies - a pharmacokinetic study in which a healthy volunteer received 150 mg tramadol hydrochloride as a single oral dose and a study in which poor and extensive CYP2D6 metabolizers received 50 mg tramadol hydrochloride as a single oral dose.