A rapid and sensitive method has been developed for the determination of the anthelmintic levamisole in plasma and tissues from man and animals. The procedure involves the extraction of the drug and its internal standard from the biological material at alkaline pH, back-extraction into sulphuric acid and re-extraction into the organic phase (heptane-isoamyl alcohol). Several extraction steps can be omitted, however, whenever the gas chromatographic background permits and some operations can be simplified using Clin ElutTM extraction tubes. The analyses were carried out by gas chromatography using a nitrogen-selective thermionic specific detector. The detection limit was 5 ng, contained in 1 ml of plasma or in 1 g of the various tissues, and recoveries were sufficiently high (79-86%). The method was applied to human plasma samples in a comprehensive bioavailability study of levamisole in healthy volunteers, and to plasma and tissues in a residue trial in cattle. The effect of the blood collection technique on the plasma levels was also studied and pointed to decreased plasma concentrations when Vacutainer tubes were used.