A total of 2,541 constituents were evaluated and prioritized for national- and regional-scale ambient monitoring of water and sediment in the United States. This prioritization was done by the U.S. Geological Survey (USGS) in preparation for the upcoming third decade (Cycle 3; 2013–23) of the National Water-Quality Assessment (NAWQA) Program. This report provides the methods used to prioritize the constituents and the results of that prioritization. Constituents were prioritized by the NAWQA National Target Analyte Strategy (NTAS) work group on the basis of available information on physical and chemical properties, observed or predicted environmental occurrence and fate, and observed or anticipated adverse effects on human health or aquatic life. Constituents were evaluated within constituent groups that were determined on the basis of physical or chemical properties or on uses or sources. Some constituents were evaluated within more than one constituent group. Although comparable objectives were used in the prioritization of constituents within the different constituent groups, differences in the availability of information accessed for each constituent group led to the development of separate prioritization approaches adapted to each constituent group to make best use of available resources. Constituents were assigned to one of three prioritization tiers: Tier 1, those having the highest priority for inclusion in ambient monitoring of water or sediment on a national or regional scale (including NAWQA Cycle 3 monitoring) on the basis of their likelihood of environmental occurrence in ambient water or sediment, or likelihood of effects on human health or aquatic life; Tier 2, those having intermediate priority for monitoring on the basis of their lower likelihood of environmental occurrence or lower likelihood of effects on human health or aquatic life; and Tier 3, those having low or no priority for monitoring on the basis of evidence of nonoccurrence or lack of effects on human health or aquatic life, or of having insufficient evidence of potential occurrence or effects to justify placement into Tier 2. Of the 1,081 constituents determined to be of highest priority for ambient monitoring (Tier 1), 602 were identified for water and 686 were identified for sediment (note that some constituents were evaluated for both water and sediment). These constituents included various types of organic compounds, trace elements and other inorganic constituents, and radionuclides. Some of these constituents are difficult to analyze, whereas others are mixtures, isomers, congeners, salts, and acids of other constituents; therefore, modifications to the list of high-priority constituents for ambient monitoring could be made on the basis of the availability of suitable methods for preparation, extraction, or analysis. An additional 1,460 constituents were placed into Tiers 2 or 3 for water or sediment, including some constituents that had been placed into Tier 1 for a different matrix; 436 constituents were placed into Tier 2 for water and 246 constituents into Tier 2 for sediment; 979 constituents were placed into Tier 3 for water and 779 constituents into Tier 3 for sediment.