Abstract This study evaluated the bioavailability of polycyclic aromatic hydrocarbons (PAHs) in Santos Bay (SB) and the adjacent Santos Continental Shelf (SCS) in Brazil. Biliary metabolites were measured in several fish species to establish a baseline for future monitoring programs. Bile samples from different species of fish were collected monthly from July to December 2005 in SB, and in August 2005 and February 2006 on SCS. Metabolite concentrations were determined using high-performance liquid chromatography with fluorescence detectors. Naphthalene, phenanthrene, and benzo[a]pyrene metabolite concentrations ranged from 24 to 810 µg g -1 of bile, 1.8 to 68 µg g -1 of bile, and below the limit of quantitation to 1.3 µg g -1 of bile, respectively. Despite its high concentrations, the levels of naphthalene metabolites were in regions of low-contamination, while benzo[a]pyrene metabolite were in the same range as those reported in moderately contaminated areas, which may indicate pyrolytic contamination by PAHs. No significant differences in the metabolite concentrations were found between the SB and the SCS samples or during the periods of collection. Future studies with a single biomonitoring species should be conducted, considering age, sex, and feeding condition of the individuals. The metabolite data presented in this study is an important baseline information for this urbanized region, which hosts several sources of contaminants.