This study investigates the mechanisms of copper tolerance of a population of black-banded rainbowfish (Melanotaenia nigrans) (Richardson). The population has been exposed to elevated copper concentrations for over 40 years, due to leachate from the Rum Jungle uranium/copper mine. At the time of collection the 96 h EC50 of exposed [E] fish was 8.3 times higher than that of reference [R] fish. The bioconcentration of 64/67Cu in fish was used to investigate the mechanism of copper tolerance in E fish. Both E and R fish were exposed to low (LCu, 30 μg Cu l-1) and elevated (ECu, 300 μg Cu l-1) copper concentrations for 24 and 48 h, respectively. Radioactivity was measured at seven or eight time points in four tissue sections: head (including gills, heart and brain), internal organs (including gastrointestinal tract, liver, kidneys and gonads), muscle and whole body. One-compartment bioconcentration models were fit to data and compared using an F-test. Copper concentrations in all tissue sections were significantly (P<0.05) less (up to 50%) in E fish compared with the respective tissue sections of R fish when exposed to both LCu and ECu. The exception was copper accumulation in the internal organs, which was not significantly different between E and R fish exposed to ECu. The mechanism of copper tolerance was concluded to be reduced copper uptake in the gills, rather than increased binding or elimination. Allozyme electrophoresis was performed to determine if genetic selection had occurred in the E fish population. Allozyme frequencies at the AAT-1 and GPI-1 loci were significantly (P<0.05) different between E and R fish. Heterozygosity was reduced in E fish compared with that of R fish. Collectively these results suggest that genetic selection may have occurred in the E fish population. Consequently, the selection of allozymes less sensitive to copper may be another mechanism of copper tolerance of E fish. This is the first study on the mechanisms of copper tolerance in a wild fish population that has been exposed to elevated copper concentrations. These findings aid the understanding of metal tolerance in fish and emphasise the importance of sample selection and its implication for toxicity testing.