Demand for groundwater is accelerating in Australia and globally. The development of guidelines to ensure ecologically sustainable use of groundwater lags behind this demand, although the broad policy and planning base for environmental water requirements is in place in Australia. Environmental water requirements in aquifers are the groundwater regimes needed to maintain or restore ecological processes and conserve biodiversity. The existing framework for environmental allocations for aquifers requires identification of groundwater dependent ecosystems and estimation of their water requirements in terms of: level (in unconfined aquifers) or pressure (in confined aquifers), discharge flux, and water quality. Of all groundwater dependent ecosystems, aquifer ecosystems are entirely groundwater dependent, but details of their biota, ecosystem processes and water requirements are the least known. This study applied the framework for determining environmental water requirements of aquifer ecosystems in a case study of the Peel Valley Alluvium, an alluvial aquifer connected to the regulated Peel River in the north-east Murray-Darling Basin, New South Wales, Australia. Eleven groundwater monitoring bores were sampled to determine whether structural indicators (abundance and taxonomic richness) of the groundwater invertebrate assemblage composition were correlated with aspects of the groundwater regime. Stygofauna (groundwater invertebrates) and physico-chemical variables were sampled quarterly in these bores from July 2006 to October 2007. Data loggers in 10 of the bores recorded groundwater level and temperature hourly for this period. Long-term stream gauge and groundwater level data were used to investigate the relationship between streamflow and groundwater regime, and to assess the degree of hydrological alteration in the river and alluvium after river regulation... A revised framework for determining environmental water requirements in alluvial aquifers is proposed which takes account of the organising principle of connectivity in delivering a compound disturbance regime, and recognizes the significance of scale and of cross-scale linkages between components of the disturbance regime. Given the current limits of distributional and taxonomic knowledge, the structural indicators of faunal assemblage composition appear inadequate to identify environmental water requirements for alluvial aquifers. The conjunctive use of functional indicators of ecosystem processes is recommended. Field-testing and further development of the framework will contribute to sustainable groundwater management, but realization of this goal relies on the collaboration of researchers, policy makers and knowledge brokers.