We have developed an improved method for the analysis of human urine for 1-hydroxypyrene (1-HOP), an accepted biomarker of polycyclic aromatic hydrocarbon uptake. This method takes advantage of commercially available 96-well format devices, which expedite sample preparation before quantitation by HPLC with fluorescence detection. In addition to improved speed of analysis, which is critical for the application of this assay in molecular epidemiology studies, the method described here uses an internal standard, 1-hydroxybenz[a]anthracene, improved sample preparation methods, and optimized HPLC and fluorescence detection conditions. The resulting method for analysis of 1-HOP is sensitive (detection limit, 0.05 pmol/mL urine), accurate (as determined by known addition of 1-HOP to urine), and precise [relative SD (RSD), 4.13%]. A longitudinal study of 1-HOP levels in the urine of 10 nonsmokers showed considerable day-to-day (mean RSD, 55.1 %) and week-to-week (mean RSD, 38.2 %) intra-individual variation, indicating the necessity for multiple sampling in studies concerned with relatively small differences in polycyclic aromatic hydrocarbon exposure.