This study was conducted to determine long-term toxic effects, on selected species of fish and invertebrates, of mine depressurization at concentrations non-lethal in acute toxicity tests. The study also includes chronic toxicity experiments, studies of sublethal effects of groundwater, and a literature review. The results of groundwater monitoring indicate that during 6 mo of storage, there was a decline in concentration of almost all chemical parameters tested. However, in contrast to previous studies, some heavy metals (iron, lead, nickel, and zinc) showed an increase in concentration with storage time. Mine depressurization groundwater was acutely toxic to the three species of invertebrates tested. The 96 h LCso for the two mayfly species, Caenis simulans and ParaZeptophZebia cornuta, was 68.75 and 64.28%, respectively. The 96 h LC so for the amphipod, Hyalella azteca, was 50%. Results of chronic toxicity tests for the three invertebrates species indicated that percent survivorship was highest in control and 3% groundwater concentrations. The mine depressurization groundwater had an inhibitor effect on the larval growth and emergence of the two mayfly species tested. The result was less obvious in the experiment with H. azteca. Cannibalism appeared to be an important factor contributing to higher mortalities in amphipods. The salinity of mine depressurization groundwater affected the osmoregulatory function of the two mayfly species. Chloride cell density was of some predictive value in determining osmoregulatory stress in the test animals. Accumulation of both Cu and Zn occurred in tissues of Caenis and Hyalella, particularly after chronic exposure to the higher concentrations of groundwater.