Studies of the effects of urbanization on stream ecosystems have usually focused on single metropolitan areas. Synthesis of the results of such studies have been useful in developing general conceptual models of the effects of urbanization, but the strength of such generalizations is enhanced by applying consistent study designs and methods to multiple metropolitan areas across large geographic scales. We summarized the results from studies of the effects of urbanization on stream ecosystems in 9 metropolitan areas across the US (Boston, Massachusetts; Raleigh, North Carolina; Atlanta, Georgia; Birmingham, Alabama; Milwaukee-Green Bay, Wisconsin; Denver, Colorado; Dallas-Fort Worth, Texas; Salt Lake City, Utah; and Portland, Oregon). These studies were conducted as part of the US Geological Survey’s National Water-Quality Assessment Program and were based on a common study design and used standard sample-collection and processing methods to facilitate comparisons among study areas. All studies included evaluations of hydrology, physical habitat, water quality, and biota (algae, macroinvertebrates, fish). Four major conclusions emerged from the studies. First, responses of hydrologic, physical-habitat, water-quality, and biotic variables to urbanization varied among metropolitan areas, except that insecticide inputs consistently increased with urbanization. Second, prior land use, primarily forest and agriculture, appeared to be the most important determinant of the response of biota to urbanization in the areas we studied. Third, little evidence was found for resistance to the effects of urbanization by macroinvertebrate assemblages, even at low levels of urbanization. Fourth, benthic macroinvertebrates have important advantages for assessing the effects of urbanization on stream ecosystems relative to algae and fishes. Overall, our results demonstrate regional differences in the effects of urbanization on stream biota and suggest additional studies to elucidate the causes of these underlying differences.