A method has been developed for the extraction and determination of sub-microgram amounts of nicotine in blood. It involves steam distillation of the nicotine followed by solvent partition and column-chromatographic clean-up. The final solution of nicotine in ethanol is injected directly on to the column of a gas-liquid chromatograph fitted with a flame-ionisation detector. The method can be used to determine down to 1 ng of nicotine, and has been applied to the measurement of nicotine levels found in the blood of smokers while smoking cigarettes.