1. In rivers affected by drought, flow regulation can further reduce flow and intensify its effects.We measured ecological responses to environmental flows, during a prolonged drought in aregulated river (Cotter River), compared with a drought affected, unregulated river (GoodradigbeeRiver) in south-eastern Australia.2. Environmental flows in the regulated Cotter River were reduced from a monthly average baseflow of 15 MLd)1 to only 5 MLd)1, which was implemented as two test flow regimes. Initially,flows were delivered in cycles of 14 days at 3 MLd)1 followed by 3 days at 14 MLd)1 and thenanother 14 days at 3 MLd)1 to make up the monthly average of 5 MLd)1. This flow regimecontinued for 6 months, after which a preliminary ecological assessment indicated deterioration inriver condition. Consequently, the flow regime was altered to a cycle of 2 MLd)1 for 28 daysfollowed by 20 MLd)1 for either 3 or 4 days. This new flow regime continued for another5 months.3. The ecological outcomes of the test flow regimes were assessed in terms of (i) the provision ofavailable habitat (wetted channel) for aquatic biota; (ii) the accumulation of periphyton; and (iii)the structure and richness of macroinvertebrate assemblages.4. Flow of 20 MLd)1 covered most of the streambed in the Cotter River, thus providing morewetted area and connectivity between habitats than flows of 2, 3 or 14 MLd)1. Depth and velocitywere always less in the Cotter River than in the unregulated Goodradigbee River. Periphytondecreased in the Cotter River during the 2 '20 MLd)1 flow regime, which combined the lowest andgreatest test flow volumes, while periphyton did not change significantly in the unregulated river.5. The reduced flow in the Cotter River resulted in fewer macroinvertebrates than expected (13)compared with unregulated Goodradigbee sites (19), although the magnitude of the differences did not depend on the test flow releases. Macroinvertebrates in the Cotter River becamenumerically dominated by Diptera and Oligochaeta, while Ephemeroptera, Plecoptera andTrichoptera decreased in abundance.6. In the Cotter River, the monthly average flow of 5 MLd)1 (exceeded 97% of the time preregulation)was insufficient to maintain the macroinvertebrate assemblages in reference condition,regardless of release patterns. However, short-term ecological objectives were achieved, such asreduced periphyton accumulation and increased habitat availability, and the environmental flowsmaintained the river's ability to recover (resilience) when higher flows returned.