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A high performance liquid chromatographic (HPLC) method for the quantification of 2-hydroxyfluorene (2-OHF) in normal human urine was established using deuterated 2-hydroxyfluorene (2-OHF-d9) as an internal standard with column-switching and fluorescence detection. The 2-OHF-d9 was synthesized by the metabolism of deuterated fluorene with cytochrome P450. The analytes were cleaned up on an ODS pre-column, via column-switching, and separated on an alkylamide-type reversed phase column. The internal standard eluted immediately prior to non-deuterated 2-OHF on the HPLC system and had nearly the same fluorescence characteristics as the non-deuterated 2-OHF. The detection limit was 0.03 nmol l(-1) (S/N = 3) and the calibration range of urine sample was from 0.2 to 50 nmol l(-1). The urine sample treatment involved enzymatic hydrolysis followed by solid phase extraction using a Sep-Pak C18 cartridge. 2-OHF was observed in the form of conjugates such as glucuronide and/or sulfate in human urine, and urinary metabolites were completely hydrolyzed for 2 h with beta-glucuronidase/aryl sulfatase. The proposed method was used to determine urinary 2-OHF in smokers and non-smokers, and showed that the urinary concentrations of 2-OHF in smokers were significantly higher than those in non-smokers (P < 0.01). Thus, the data suggest that urinary 2-OHF might be a sensitive and specific biological marker for the assessment of the exposure to polycyclic aromatic hydrocarbons.