The recent interest in ecosystem management has brought an awareness of the need for an appropriate spatial framework for effectively structuring ecosystem management as well as the research, inventory, and monitoring of ecosystems and their components. This paper addresses the complexity of the task of defining ecological regions as well as the concept of regional reference sites, and provides an explanation of the development of the hierarchical ecoregion framework within the U.S. Environmental Protection Agency. Comparisons are made with other frameworks such as watersheds or basins. Also covered are the processes by which ecoregions are refined, subregions are defined, and sets of reference sites are selected via collaborative projects involving state and federal resource managers and EPA geographers. Although development of the ecoregion/reference site framework has to date been largely driven by the needs of state resource management agencies for developing biological criteria, and for setting water quality standards and lake management goals, support has grown for structuring biological risk assessment and ecosystem management in general.