The effect of pH on the acute toxicity of Cu to barley (Hordeum vulgare) root elongation was investigated in solution culture. The results showed that the median effective concentrations (EC50s; i.e., the concentration that reduced root elongation by 50% based on free Cu2+ activity) were not significantly different in the low-pH range from 4.5 to 6.5, but in the high-pH range from 7.0 to 8.0, a significant effect of pH on EC50s was found. The nonlinear relationship between EC50 and H+ activity in the present study indicated that the increased toxicity with increasing pH in solution may not be caused by decreasing H+ competition. When we take account of CuOH+ activities, a good linear relationship (r2 > 0.97) between the ratio of CuOH+ activity to free Cu2+ activity and acute Cu toxicity to barley root elongation was achieved, which indicated that the observed toxicity in the high-pH range may be caused by CuOH+ plus free Cu2+ in solution. Linear-regression analysis suggested CuOH+ had a greater binding affinity than Cu2+ at the biotic ligand sites. The logistic dose–response curve showed that expressing the Cu dose as Cu2+ + 2.92·CuOH+ improved the data fit significantly compared to consideration of the free Cu2+ activity only. Thus, our results suggest CuOH+ was highly toxic to barley root elongation. The enhanced toxicity of CuOH+ therefore needs to be considered when modeling the effect of pH on Cu toxicity to barley for exposures having pH greater than 6.5.