Integrated hydrological and biological data are crucial to understanding the ecological dynamics of streamflow intermittency. Our objectives were to determine the hydrologic characteristics that drive biological community structure along an intermittency gradient, and to identify biological signatures of flow intermittency. We sampled macroinvertebrate assemblages in 21 southern California streams that varied primarily in hydrologic permanence. Sampling sites were located at stream gages in basins with minimal human influence. Mean annual zero-flow days ranged from 0-200 based on daily flow records ranging from 11-100 years in length. Biological assemblage composition was evaluated for associations with several hundred streamflow metrics calculated over long- (decades) and short-term (antecedent 365 days) temporal windows. We identify several streamflow characteristics that were the most influential predictors of benthic assemblage structure. We also report the taxonomic and trait-based biological indicators representative of various degrees of intermittency.