A technique has been developed to separate a mixture of the twelve isomeric phenols of benzo(a)pyrene by using High-Performance Liquid Chromatography with fluorescence detection and various sequences of methanol/water gradients. Detection by fluorescence has increased the sensitivity of the method when compared with UV detection. The effects of flow rate, temperature, and various types of gradients on the separation of the isomeric phenols are discussed. It has been found that under the conditions normally employed to separate these phenols from other metabolites of benzo(a)pyrene, several of the phenols are not separated possibly leading to incorrect identification as well as quantitation. Diols and diones of benzo(a)pyrene are also separated by this procedure.