Tramadol, an analgesic agent, and its main metabolites O-desmethyltramadol (M1), N-desmethyltramadol (M2) and O,N-didesmethyltramadol (M5) were determined simultaneously in human plasma, saliva and urine by a rapid and specific HPLC method. The sample preparation was a simple, one-step, extraction with ethyl acetate. Chromatographic separation was achieved with a Chromolith Performance RP-18e 100 mm x 4.6 mm column, using a mixture of methanol:water (19:81, v/v) adjusted to pH 2.5 by phosphoric acid, in an isocratic mode at flow rate of 2 ml/min. Fluorescence detection (lambda(ex) 200 nm/lambda(em) 301 nm) was used. The calibration curves were linear (r(2)>0.996) in the concentration ranges in plasma, saliva and urine. The lower limit of quantification was 2.5 ng/ml for all compounds. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were acceptable in all analyzed body fluids The developed procedure was applied to assess the pharmacokinetics of tramadol and its main metabolites following administration of 100mg single oral dose of tramadol to healthy volunteers.