Toxicity of copper to Daphnia magna in artificial medium, artificial medium plus algae, and natural water from Hamilton Harbour and Lake Ontario was determined, with and without added Tris, in order to test the assumption that free cupric ion concentrations are the same in equivalently toxic media even when total copper concentrations vary. Free cupric ion concentrations were calculated from the increase in the total copper concentration tolerated after Tris addition and from cupric ion electrode measurements. Copper toxicity was greatest in inorganic medium, lowest in inorganic medium plus algae and in Hamilton Harbour water, and intermediate in lake water. However, after Tris addition, toxicity was greatest in lake water and lowest in inorganic medium and inorganic medium plus algae. Both the bioassay and electrode data indicate that free cupric ion concentrations differ in different test waters when toxicity is the same. Free metal concentrations do not, therefore, provide a good measure of copper toxicity for Daphnia in natural waters.