While it is generally accepted that water hardness affects copper toxicity, the major ions that contribute to water hardness (calcium [Ca] and magnesium [Mg]) may affect copper toxicity differently. This is important because the Ca:Mg ratio in standard laboratory-reconstituted waters often differs from the ratio in natural surface waters. Copper toxicity was assessed for five different aquatic species: rainbow trout (RBT), fathead minnow (FHM), Ceriodaphnia dubia, Daphnia magna, and an amphipod (Gammarus sp.) under different Ca:Mg ratios (4:0, 3:1, 1:1, 1:3, and 1:4 mass basis) at a common hardness (180 mg/L as CaCO3) and alkalinity (120 mg/L as CaCO3). Copper toxicity increased at lower Ca:Mg ratios for RBT but increased at higher Ca:Mg ratios for D. magna. Fathead minnows (<24 h old) were more sensitive to copper in 1:1 Ca:Mg waters compared to 3:1 Ca:Mg waters. The toxicity of copper did not vary under different Ca:Mg ratios for Gammarus sp., C. dubia, and 28-d-old FHM. The effect of Ca:Mg ratios on copper toxicity changed for D. magna in softer water (90 mg/L as CaCO3) compared with hard water studies.