A high-performance liquid chromatography (HPLC)/fluorescence method for quantitative analysis of 1-hydroxypyrene in urine was developed. The method validation analysis showed the method to be in analytical control. No significant systematical errors could be demonstrated. The entire run time of chromatography was 10 min using isocratic elution (acetonitrile-water, 70:30), and the retention time for 1-hydroxypyrene was 3.5 min. The short run time in combination with the low limit detection (1.37 nmol/L) makes the method potentially applicable for surveillance of pyrene exposure in work environments. The developed method is presently used for measurement of 1-hydroxypyrene in urine samples from workers exposed to a low airborne level of polycyclic aromatic hydrocarbons, generally less than 25 micrograms/m3. The urine samples of exposed workers (n = 122) showed a range of 1-hydroxypyrene from the limit of detection (LD) up to 39 nmol/L, whereas unexposed control individuals (n = 108) showed a range from LD to 3.3 nmol/L.