The effect of acclimation time, habituation period, and final freshwater ion composition on the survival of freshwater-acclimated Pacific white shrimp Litopenaeus vannamei postlarvae was investigated. During each of three experiments, shrimp were acclimated from 30 ppt to freshwater (<1 ppt TDS) utilizing various acclimation times (32, 40, 48, 72 h) with a constant or variable rate of salinity reduction. Shrimp were then held at the final acclimation salinity for 0, 1 or 2 days (habituation period) before being transferred to challenge ion treatment solutions. Ion treatment solutions derived from chloride-based chemicals were of the same total ion concentration, but either strongly monovalent or strongly divalent. An acclimation time of 72 h compared to 48 h, with no habituation period, increased shrimp survival by 27%. A 1-day habituation period compared to no habituation after 48-h acclimation also increased mean shrimp survivals by 27%. Decreasing acclimation time at higher salinities (i.e., 30 down to 1 ppt) and increasing acclimation time at lower salinities (<1 ppt) allowed successful freshwater acclimation of shrimp within 32 h. This demonstrates a critical period for freshwater acclimation of marine shrimp at lower salinities. The longer freshwater acclimation time and habituation period probably allowed shrimp to equalize hemolymph ions before transfer to ionically unbalanced challenge solutions. The survival of freshwater-acclimated shrimp can be improved by either extending the acclimation time from 48 to 72 h or providing a habituation period of 2 days after 48-h acclimation. However, growth at these ion concentrations still needs to be verified.