Many ambient water quality criteria established to protect aquatic life from acute toxicity are calculated using a procedure described in the U.S. Environmental Protection Agency’s 1985 guidelines. The procedure yields a final acute value (FAV) from acceptable LC50 or EC50 concentrations that is a single point, deterministic estimate of the concentration of a chemical substance that will protect 95% of aquatic species from > 50% mortality or other acute toxic effects. However, because of variation and uncertainty associated with toxicity test results, uncertainty in the estimated FAV exists that is not accounted for by the 1985 guidelines procedure. Here, Monte Carlo analysis is used to characterize this uncertainty. The analysis uses copper EC50 values adjusted for differences in test water chemistry obtained from USEPA’s final freshwater copper criteria guidance published in 2007. Additional Monte Carlo simulations illustrate the effects on simulated FAV distributions of testing with fewer species and fewer replicate tests. The deterministic procedure yields an FAV of 4.68 ug/L for the complete data set. By comparison, three replicate Monte Carlo simulations yielded mean FAVs of 4.66 ug/L. The distribution of calculated FAVs ranged from 3.59 ug/L to 5.92 ug/L with 5th and 95th percentiles of 4.14 ug/L and 5.20 ug/L, respectively. Reducing the number of tested genera from 27 to 8 (the minimum required by the 1985 guidelines) and setting the number of tests per species equal to 3 yielded a range of simulated FAV values between 0.38 ug/L and 13.1 ug/L compared to a deterministic estimate of 2.80 ug/L. The 5th and 95th percentiles were 1.22 ug/L to 6.18 ug/L. Results of this study indicate that Monte Carlo analysis can be used to improve the understanding and communication of uncertainty associated with water quality criteria. This may benefit the development, revision and application of these criteria in the future.