Trans-3'-hydroxycotinine was found to be a main metabolite of nicotine in smokers. It is the most abundant among all nicotine metabolites, if the total urine is collected over 7 consecutive days of smoking and 5 following days without smoking. Its steady-state blood plasma concentration on the 6th day of smoking is second only to cotinine. In the experiments nine habitual smokers smoked 19 cigarettes per day on 7 days of smoking. Three runs were carried out with three types of cigarettes which differed in mainstream nicotine. After oral administration of 30 mg trans-3'-hydroxycotinine, the cumulative excretion rates of two subjects were 81% and 93% of the unchanged compound, respectively.