While immunoassays have been extensively applied to evaluate environmental contamination, to date they have rarely been used for the analysis of biological fluids outside of human medicine. These media are important because pollutants such as polycyclic aromatic hydrocarbons (PAHs) and their metabolites become concentrated in tissues, body fluids, and excreta, thereby offering a measure of exposure to biologically available contaminants. Such analyses also provide a nondestructive tool for monitoring exposure. Crabs (Carcinus maenas) were exposed to phenanthrene and pyrene (separately) at concentrations ranging from 0 to 200 μg L-1. After 48 h, urine samples were taken and analyzed by immunoassay and UV-fluorescence spectrophotometry. Urinary levels (calibrated against hydroxylated metabolites) proved to be dose dependent for both compounds, and good agreement was demonstrated between the immunoassay and the fluorescence techniques. The cross reactivity of the immunochemical technique (ELISA) for pyrene and hydroxy-metabolites was lower than for phenanthrene. HPLC analyses demonstrated that urine from the crabs exposed to pyrene contained mainly conjugate PAH metabolites whose concentrations (the sum of the four main pyrene metabolites/conjugates) showed very good agreement with the ELISA (r2 > 0.94) and fluorescence (r2 > 0.91) data. Environmental samples were also analyzed by ELISA and UV-fluorescence, and both techniques detected PAH (mainly petrogenic) contamination in the urine samples from a polluted harbor. These data demonstrate the potential of urine analyses by ELISA and UV-fluorescence to measure exposure of crabs to PAH.