Nine male smokers smoked three types of cigarettes that differed in mainstream nicotine. The experiment was carried out in three runs, each consisting of 7 d when smoking was allowed and 5 d when it was not allowed. Urine fractions were collected throughout the whole period of the experiments. As assumed, nicotine and four of its known metabolites, i.e., cotinine, nicotine-N'-oxide, nornicotine, and N-methyl-nicotinum ions were found in the urine. In addition, trans-3'-hydroxycotinine was determined. The amount of this metabolite was well in excess of those of nicotine and cotinine in the urine. The serum concentration trans-3'-hydroxycotinine was found to be second to serum cotinine.