Instream limestone sand application is used at many sites in Pennsylvania to neutralize acidic stream water resulting from acid deposition. However, questions remain about the effectiveness of limestone sand in the treatment of acid waters, such as reduced contact time at high flow, remobilization of aluminum, and adverse effects on macroinvertebrates. A 1-year evaluation of limestone sand application to Bear Run, an acidic stream in southwestern Pennsylvania, was begun in 1999. Another nearby acid stream, Linn Run, served as a control. Water quality during baseflow and episodic acidification events, along with fish and macroinvertebrates, were monitored to evaluate impacts of the sand application. Hydrogen ion (H+) concentration and total dissolved aluminum (TDA) were significantly reduced, and acid neutralizing capacity was significantly increased downstream of the limestone sand application compared with the upstream site on Bear Run. These parameters at the downstream sites were also different (∝ 0.10) from the comparable sites on the control stream. TDA and hydrogen ion concentrations were significantly decreased (∝ 0.10) compared with concentrations before the sand application to Bear Run but not on the control stream. No fish were present upstream of the sand application site, and only a few fish were found downstream at the mouth of Bear Run. Standing crop, number of taxa, and Shannon-Weaver diversity index values indicated that macroinvertebrate populations were negatively impacted 300 m downstream from the sand application and improved at the site 1,600 m downstream. Although water quality was improved on Bear Run, benefits to macroinvertebrates depended on downstream location, and fish populations did not show improvement.