Recent studies of quantitative and qualitative aspects of nicotine metabolism, and the use of metabolic data for estimating nicotine intake in humans are discussed. Previously reported methodology for determining nicotine bioavailability has been improved by using stable isotope-labeled nicotine administered intravenously. Combined gas chromatography-mass spectrometry with selected ion monitoring has been used to simultaneously determine concentrations of isotopically-labeled nicotine and tobacco-derived nicotine in blood. Nicotine intake from cigarette smoking was estimated from clearance of labeled nicotine and blood concentrations of tobacco-derived nicotine. Progress in elucidating the metabolic profile of nicotine in humans, and the use of nicotine metabolites as markers of tobacco smoke consumption is summarized.