An enzyme immunoassay was developed for the simultaneous detection of different pyrethroid insecticides. Several polyclonal antisera were raised in rabbits using 1R trans-permethric acid coupled to bovine serum albumin (BSA) as immunogen. Antiserum D12 recognized permethrin, cyfluthrin, cypermethrin. S-bioallethrin and the metabolite 1R trans-permethric acid with similar affinities. 50 % inhibition (50 % B/B0) for permethrin occurred at 8 μg/L; linearity ranged between 0.4 and 74 μg/L. The antisera D5 and D7 were obtained from preceding blood samples, and the antibodies showed different cross-reactivities, revealing the path of affinity maturation of the antiserum.