A method using a molecularly imprinted polymer (MIP) as the selective sorbent for solid-phase extraction (SPE) has been developed. Its application to the assay of hairy nicotine level among smokers and non-smokers with high-performance liquid chromatography (HPLC) and evaluation of exposures to the environmental tobacco smoke (ETS) were validated. The MIP was synthesized using nicotine as the template molecule and methacrylic acid (MAA) as the functional monomer. This MIP-SPE method provided inherent selectivity and a sensitive response to nicotine with a detection limit of 0.2 ng/ml hair at a signal-to-noise ratio of 3:1 and the limit of quantification was 0.5 ng/ml. The linearity was assessed in the range of 0.5-80 ng/ml hair, with a coefficient (r(2)) greater than 0.987. The amounts of nicotine determined in smokers and non-smokers hair were in the range of 5.1-69.5 ng/mg hair and 0.50-9.3 ng/mg hair, respectively. The reported measures of ETS exposure were significantly associated with hairy nicotine levels. This assay of nicotine in hair using MISPE provided a very selective and reliable method for the evaluation of the exposure to tobacco smoke.