The effects of changes in the landscape and alteration of natural flow process on aquatic macroinvertebrate assemblages were investigated in 67 small-to-medium sized (15–526 km2) upland streams in the northeastern United States. Environmental characteristics that were found to be important in determining macroinvertebrate-assemblage composition include urbanization and concomitant changes in natural streamflow patterns. In particular, hydrologic attributes accounted for a significant proportion of the variability and were important in driving modifications to assemblage structure after natural environmental variability was extracted. For example, mean April flow accounted for the greatest amount of assemblage variability in any single multiple linear regression (MLR) model (65%) and duration of high flows accounted for a significant portion of the assemblage variability in the five, four and one-variable models (25, 26, and 23%, respectively). Seasonal predictability of low flow consistently accounted for a significant proportion of the assemblage variability in all but the two-variable (MLR) model. Significant (p < 0·05) bivariate flow–ecology response relations were established, especially for hydrologic measures that account for the frequency, duration, and magnitude of flow events, and these relations generally followed increasing or decreasing trends that would be expected given changes in stream hydrology. This study demonstrates that there are likely specific negative consequences to stream biotic integrity in northeastern streams as the result of hydrologic alteration associated with basin urbanization. Understanding the relations between hydrologic modification and aquatic assemblages will help efforts to set sustainable flow standards for protection of aquatic assemblages while providing water for human needs. Published in 2009 by John Wiley & Sons, Ltd.