Measurement of plasma cotinine, the major metabolite of nicotine, is usually done to determine nicotine-intake in smokers. Cotinine is used instead of nicotine because it has a much longer half-life than the mother substance and its plasma concentrations are therefore less dependent on the exact times of blood sampling. However, the linearity of the relationship between nicotine-intake and cotinine level in plasma has never been proven. Therefore cotinine was measured in 6 healthy volunteers infused over 4 days with several doses of nicotine i.v. up to 480 micrograms/kg/day. Cotinine concentrations in plasma were shown to be linearly and directly related to nicotine intake. The concentration of cotinine showed little variation during and for up to 2 h after the last dose of nicotine. Therefore, cotinine can be used as an epidemiological marker of nicotine intake if it is measured around the time of the last cigarette of the day.