Polyclonal rabbit anticotinine antiserum, which can be used for biomonitoring nicotine uptake by the determination of cotinine in body fluids, was checked by a competitive ELISA for its cross-reactivity with nine nicotine metabolites. The highest percentage of relative cross-reactivity (about 30%) was observed with trans-3'-hydroxycotinine, a metabolite which is known to be excreted in 3-fold higher amounts than cotinine in the urine of human smokers. Therefore, it is possible that cotinine determinations performed by immunochemical methods--especially in urine--may yield overestimated cotinine concentrations.