Regulated rivers in the United States are required to support human water uses while preserving aquatic ecosystems. However, the effectiveness of hydropower license requirements nationwide has not been demonstrated. One requirement that has become more common is "run-of-river" (ROR) operation, which restores a natural flow regime. It is widely believed that ROR requirements (1) are mandated to protect aquatic biota, (2) decrease hydropower generation per unit flow, and (3) decrease energy revenue. We tested these three assumptions by reviewing hydropower projects with license-mandated changes from peaking to ROR operation. We found that ROR operation was often prescribed in states with strong water-quality certification requirements and migratory fish species. Although benefits to aquatic resources were frequently cited, changes were often motivated by other considerations. After controlling for climate, the overall change in annual generation efficiency across projects because of the change in operation was not significant. However, significant decreases were detected at one quarter of individual hydropower projects. As expected, we observed a decrease in flow during peak demand at 7 of 10 projects. At the remaining projects, diurnal fluctuations actually increased because of operation of upstream storage projects. The economic implications of these results, including both producer costs and ecologic benefits, are discussed. We conclude that regional-scale studies of hydropower regulation, such as this one, are long overdue. Public dissemination of flow data, license provisions, and monitoring data by way of on-line access would facilitate regional policy analysis while increasing regulatory transparency and providing feedback to decision makers.