Tobacco smoke, certain foods, and diesel exhaust are known to contain polycyclic aromatic hydrocarbons (PAHs). Historically, 1-hydroxypyrene (1-OHP), a metabolite of pyrene, has been the most commonly used biomarker of exposure to PAHs. More extensive analytical methods covering multiple urinary PAH metabolites, however, are needed to adequately assess human exposure to a mixture of PAHs. Analysis of replicate single low dose urine samples for many hydroxy PAHs with quantitative recovery and high precision requires effective enzymatic hydro1ysis, efficient sample clean up, high resolution chromatography, selective detection, sensitive response, and fast turn-around time. During the past five years, several reports (to different degrees) have appeared that address with varying success these issues. This presentation will focus on the analysis of 1-OHP, 3-hydroxyphenanthrene (3-OHPh), and 2-hydroxybenz[c]phenanthrene (2-OHBcPh) in urine. Optimized liquid parameters for solid phase extraction along with the merits of quantitative analysis using liquid chromatography via fluorescence versus tandem mass spectrometric electrospray detection will be described. Replicate percent recoveries via external and internal standard calibration for each of the three analytes will be reported. The advantage and disadvantage of single versus multiple internal standard calibrations as it relates to multiple urinary PAH metabolite determination will be discussed.