Brown trout (Salmo trutta) were captured from a headwater stream contaminated with moth‐proofing agents. Synthetic pyrethroid residues within the tissues were obtained by ultrasonic extraction and examined by gas chromatography‐mass spectrometry operated in the negative ion chemical ionization mode (GC/NCI‐MS). Both permethrin and cyfluthrin had been bioaccumulated by the fish and the mean concentrations ( ± standard deviation) recorded were 2046 ( ± 203) μg kg‐1and 25.4 ( ± 46.9) μg kg‐1respectively. The concentrations of these pyrethroids are the highest recorded for freshwater fish captured from a natural ecosystem. At the time of sampling, permethrin could be detected within the stream water (0.034 μg l‐1) while cyfluthrin could not. A mean relative bioconcentration factor of approximately 60 000 was derived for the brown trout which was also greater than any previously determined for freshwater fish. The fish tissue extracts were also analyzed through use of an ELISA for permethrin. The ELISA reported consistently lower concentrations than those obtained by GC/NCI‐MS. This was attributed to analyte losses during preparation of the sample extracts in a form suitable for incorporation into the ELISA. The concentrations obtained by ELISA were significantly correlated with those obtained by GC/NCI‐MS (R2= 0.985, n = 5). Overall, the ELISA appeared to be suitable for the detection of permethrin in fish when present at concentrations in excess of the maximum residue level suggested for many foods by the Commission of the European Economic Community.