Naphthenic acids (NAs) are the most water-soluble organic components found in the Athabasca oil sands in Alberta, Canada, and these acids are released into aqueous tailing waters as a result of bitumen extraction. Although the toxicity of NAs to fish is well known, there has been no method available to estimate NAs concentrations in fish. This paper describes a newly developed analytical method using single ion monitoring gas chromatography–mass spectrometry (GC–MS) to measure NAs in fish, down to concentrations of ∼0.1 mg kg−1 of fish flesh. This method was used to measure the uptake and depuration of commercial NAs in laboratory experiments. Exposure of rainbow trout (Oncorhynchus mykiss) to 3 mg NAs l−1 for 9 d gave a bioconcentration factor of ∼2 at pH 8.2. Within 1 d after the fish were transferred to NAs-free water, about 95% of the NAs were depurated. In addition, the analytical method was used to determine if NAs were present in four species of wild fish – northern pike (Esox lucius), lake whitefish (Coregonus clupeaformis), white sucker (Catostomus commersoni), walleye (Sander vitreus) – collected from near the oil sands. Flesh samples from 23 wild fish were analyzed, and 18 of these had no detectable NAs. Four fish (one of each species) contained NAs at concentrations from 0.2 to 2.8 mg kg−1. The GC–MS results from one wild fish presented a unique problem. However, with additional work it was concluded that the NAs concentration in this fish was <0.1 mg kg−1.