Copper-sensitive bacteria were isolated from a tropical river water sample and used to develop a simple, inexpensive bioassay for the determination of copper bioavailability in freshwaters. The 48-h bioassay, which has an EC50 for inorganic copper of 7.1 g / L, involved addition of copper-sensitive bacteria to filtered water samples amended with growth-stimulating nutrients and measurement of growth. Matrix-matched control solutions were prepared by adding the metal complexing agent EDTA to a portion of each sample tested. In order to optimize sensitivity of the bioassay, a nutrient medium was developed, which stimulated bacterial growth, but did not appreciably alter copper speciation of the sample. This necessitated omission of many organic compounds commonly incorporated in growth media because of their ability to complex copper. Performance of the bacterial bioassay was evaluated on freshwater samples from tropical and temperate river systems. Bacterial 48-h EC15 values ranged from 5.0 to 25.0 g Cu / L and were much higher than the observed EC15 value of 2.8 ( ) ( g / L for inorganic copper in deionized water no organic matter present . A linear correlation r = 0.93, ) p ( 0.005 was observed between the measured EC15 values and the copper complexation capacities of river water samples determined by anodic stripping voltammetry. This confirmed the role of natural organic matter in ameliorating copper toxicity. Results of this study provide further evidence that the bioavailability of copper in freshwaters is significantly reduced in the presence of natural organic matter. A generic protocol for development of metal-sensitive bacterial bioassays was developed as part of the work.