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Restoring the ecological integrity of regulated large rivers necessitates characterizing the natural flow regime. We applied ‘Indicators of Hydrologic Alteration’ to assess the natural range of variation of the Missouri River’s flow regime at 11 locations before (1929–1948) and after (1967–1996) mainstem impoundment. The 3768 km long Missouri River was divided into three sections: upper basin least-altered from flow regulation, including the lower Yellowstone River; middle basin inter-reservoir, and lower basin channelized. Flow regulation was associated with a reduction in magnitude and duration of the annual flood pulse, an increase in magnitude and duration of annual discharge minima, a reduction in frequency of annual low-flow pulses, earlier timing of March–October low-flow pulses, and a general increase in frequency of flow reversals with a reduction in the rate of change in river flows. Hydrologic alterations were smallest at two least-altered upper-basin sites and most frequent and severe in inter-reservoir and upper-channelized river sections. The influence of reservoir operations on depressing the annual flood pulse was partially offset by tributary inflow in the lower 600 km of river. Reservoir operations could be modified to more closely approximate the 1929–1948 flow regime to establish a simulated natural riverine ecosystem. For inter-reservoir and upper channelized-river sections, we recommend periodic controlled flooding through managed reservoir releases during June and July; increased magnitude, frequency and duration of annual high-flow pulses; and increased annual rates of hydrograph rises and falls. All of the regulated Missouri River would benefit from reduced reservoir discharges during August–February, modified timing of reservoir releases and a reduced number of annual hydrograph reversals. Assessment of ecological responses to a reregulation of Missouri River flows that more closely approximates the natural flow regime should then be used in an adaptive fashion to further adjust reservoir operations.