Three monoclonal antibodies (mab) for the determination of the pyrethroid insecticide allethrin were generated using a trans-permethric acid-bovine serum albumin conjugate as immunogen. Spleen cells of immunized BALB/c mice were fused with myeloma cells applying polyethylene glycol. Hybridoma cells were selected by HAT selection and screened for cell lines producing mab. Three stable cell lines could be isolated for further characterization and development of an indirect competitive enzyme immunoassay. The mab 3/B4-1/F8 showed a preference for S-bioallethrin, the most efficient insecticidal isomer of allethrin. The detection range lay between 1 μg/L and 250 μg/L; 50 % inhibition occurred at 46 μg/L. Cross-reactivities were 43 % for the natural pyrethrins. The mab 1/E2-5/B5 and 1/A5-7/C10 preferred bioallethrin, which is a mixture of two allethrin enantiomers. 50 % inhibition was found at 60 μg/L or 54 μg/L, respectively. The tests ranged from 9 μg/L to 170 μg/L (1/E2-5/B5) and from 6 μg/L to 160 μg/L (1/A5-7/C10). Cross-reactivities occurred for phenothrin, resmethrin and bioresmethrin.