Abstract   The objective of the present study was to establish a culture of Hyalella azteca that could be used for laboratory toxicity testing in low–ionic strength waters with electrical conductivities of <200 μS/cm. A wild strain of H. azteca was collected from Twin Lake, a small seepage lake with an electrical conductivity of 81 ± 27 μS/cm located on the property of Chalk River Laboratories in Chalk River, Canada. To determine the minimum aqueous ion requirements for an optimal culturing medium for the Twin Lake strain, Twin Lake was monitored for water quality and ionic content over 4 yr. Water quality parameters were averaged and used to formulate a medium containing NaHCO3, CaCl2, MgSO4, KCl, NaBr, NaF, and LiCl, with an electrical conductivity of 89 ± 3 μS/cm. By evaluating survival and reproduction, it was concluded that this artificial medium promoted survival and supported reproduction (10 ± 4 neonates/female/wk) of the Twin Lake amphipod. The Twin Lake strain of H. azteca can, therefore, be maintained in laboratory settings, and this allows for toxicity testing to be conducted on low–ionic strength waters. Environ Toxicol Chem 2019;38:585–590. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.