Simultaneous HPLC determination of the analgetic agent tramadol, its major pharmacodynamically active metabolite (O-desmethyltramadol) in human plasma is described. Simple methods for the preparation of the standard of the above-mentioned tramadol metabolite and N1,N1-dimethylsulfanilamide (used as the internal standard) are also presented. The analytical procedure involved a simple liquid-liquid extraction of the analytes from the plasma under the conditions described previously. HPLC analysis was performed on a 250x4 mm chromatographic column with LiChrospher 60 RP-selectB 5-microm (Merck) and consists of an analytical period where the mobile phase acetonitrile-0.01 M phosphate buffer, pH 2.8 (3:7, v/v) was used, and of a subsequent wash-out period where the plasmatic ballast compounds were eluted from the column using acetonitrile-ultra-high-quality water (8:2, v/v). The whole analysis, including the equilibration preceding the initial analytical conditions lasted 19 min. Fluorescence detection (lambda(ex) 202 nm/lambda(em) 296 nm for tramadol and its metabolite, lambda(ex) 264 nm/lambda(em) 344 nm for N1,N1-dimethylsulfanilamide) was used. The validated analytical method was applied to pharmacokinetic studies of tramadol in human volunteers.