In 1991, the U.S. Geological Survey (USGS) began a study of more than 50 major river basins across the Nation as part of the National Water-Quality Assessment (NAWQA) project of the National Water-Quality Program. One of the major goals of the NAWQA project is to determine how water-quality and ecological conditions change over time. To support that goal, long-term consistent and comparable ecological monitoring has been conducted on streams and rivers throughout the Nation. Fish, invertebrate, and diatom data collected as part of the NAWQA program were retrieved from the USGS Aquatic Bioassessment database for use in trend analysis. Ultimately, these data will provide insight into how natural features and human activities have contributed to changes in ecological condition over time in the Nation’s streams and rivers. This USGS data release contains all of the input and output files necessary to reproduce the results of the ecological trend analysis described in the associated U.S. Geological Survey Scientific Investigations Report. Data preparation for input to the model is also fully described in the above mentioned report.