Polyclonal antisera were produced in rabbits against two different synthetic immunogens, one of which incorporated 3‐phenoxybenzoic acid (PBA) while the other contained dichlorovinyl cyclopropane carboxylate (CPA). The immunogens were constructed such that the hapten was coupled to the carrier protein through a peptide bond to a six carbon spacing group (6‐amino hexanoic acid, 6‐AHA). Both the anti‐PBA and anti‐CPA antisera obtained were able to detect permethrin when used in an indirect competitive enzyme‐linked immunosorbent assay (IC‐ELISA) format. The detection limits typically obtained with both antisera were 10 mg l‐1with 50% inhibition of antibody binding (ho) at 100mg l‐1. Cross‐reactivity with the pyrethroids cyfluthrin, phenothrin and deltamethrin was observed for both antisera, the degree of which was related to the structural similarity of the compound to the immunizing hapten. Further development of the immunoassay for permethrin was examined through use of the anti‐PBA antiserum. Assay performance was improved by negative immunoaffinity chromatog‐raphy of the anti‐PBA antiserum, in which antibodies directed against the six carbon spacing group were removed. In conjunction with an avidin‐biotin amplification step, typical detection limits were 1 mg l‐1with an I50value of 15 mg l‐1. Assay performance was considerably enhanced by use of a microtitre plate coating antigen which possessed a four carbon spacing group between the hapten and carrier protein. The hapten was also coupled to the spacing group through an ester bond. Typical detection limits for permethrin were 0.5μg l‐1, with an I50value of 1 mg l‐1. This assay was also unaffected by the inclusion of methanol at concentrations of up to 10% by volume. The study indicated the potential usefulness of antibodies raised against compounds which mimic moieties present within larger hapten molecules (anti‐hapten mimic antibodies), particularly where the target analyte is not amenable to direct conjugation to a carrier protein.