Polycyclic aromatic hydrocarbons (PAH) are products of the incomplete combustion of organic materials, and they occur ubiquitously in the environment. They are also present in tobacco smoke. Some PAH have been classified as carcinogens; therefore, it is important to develop and assess suitable biomarkers for PAH exposure. A high-performance liquid chromatographic method with fluorescence detection was developed to determine 1- and 2-hydroxynaphthalene (1- and 2-OH-Nap), 2-hydroxyfluorene (2-OH-Flu), 2-/3-hydroxyphenanthrene (2-/3-OH-Phe), 1-/9-hydroxyphenanthrene (1-/9-OH-Phe), and 1-hydroxypyrene (1-OH-Pyr) in human urine. The method is sensitive (LOQ ranging from 0.01 ng/mL for 1-OH-Pyr to 1 ng/mL for the naphthols), precise (interday precision ranging from 1.4 to 6.9%), and accurate (97-106%). The method was applied to 108 urine samples from 25 nonsmokers and 83 smokers. Smokers excreted significantly higher amounts of 1-OH-Nap (16.1 vs. 2.9 microg/24 h), 2-OH-Nap (20.9 vs. 9.7 microg/24 h), 2-OH-Flu (1.87 vs. 0.75 microg/24 h), 2-/3-OH-Phe (0.73 vs. 0.50 microg/24 h), 1-/9-OH-Phe (0.66 vs. 0.35 microg/24 h), and 1-OH-Pyr (0.36 vs. 0.20 microg/24 h) compared to nonsmokers. In conclusion, the method is suitable for discriminating PAH exposure between different ISO tar yield cigarette smokers, and it may be applicable in evaluating future potential reduced exposure tobacco products.