In Europe, a limit value of 10ng L21 was set by the European Commission for benzo[a]pyrene(B[a]P) in water intended for human consumption (Council Directive 98/83/EC) and, therefore,sensitive and reliable methods are needed to evaluate its presence. We report here on thedevelopment of a highly sensitive indirect competitive ELISA for the detection of B[a]P in potablewater. Fourteen monoclonal antibodies were generated in mice using novel B[a]P derivatives. Theimmunoassay with the least interference and the best sensitivity was optimized and characterized.As co-solvent, ten percent methanol (v/v) was determined as the optimum concentration for B[a]Psolubilization for use with the developed ELISA. With the purified antibody (clone 22F12) theaverage IC50 for B[a]P and corresponding detection limit at a signal:noise (S/N) ratio of 3 was65 ng L21 and 24 ng L21, respectively. From the 16 EPA-designated PAHs, only chrysene,indeno[1,2,3-cd]pyrene, and benzo[b]fluoranthene showed a cross-reactivity (CR) higher than20%. No CR was observed for two- and three-ringed aromatics as well as dibenz[ah]anthraceneand benzo[ghi]perylene. The effect of pH value (range 6.5-9.5), ionic strength (specific electricconductivity 1 mS cm21–2.5mS cm21), and inorganic ions (sodium, copper, iron, aluminium,manganese, chloride, sulfate, nitrate, and nitrite at maximum permissible levels according to theCouncil Directive) on both signal and sensitivity of the ELISA was studied. No significantinfluence of these parameters on the ELISA competition curve was found. We suggest that theoptimized ELISA can be used to monitor potable water samples without previous extraction fromthe samples. The assay should facilitate the cleanup of B[a]P contaminated sites where B[a]P levelsfall close to the limit value of the new drinking water directive.