Exposure to arsenic is known to cause adverse effects in aquatic biota and wildlife and is of major concern to human health. Although numerous studies have investigated the toxicity of arsenic, little is known about the effects of acquired tolerance on arsenic accumulation and toxicity outside of cell culture models. Accordingly, studies were conducted on the estuarine fish, Fundulus heteroclitus, that were preexposed to nontoxic concentrations of arsenic (as sodium arsenite; 0.7 and 106 μmol As/L) for 96 h or naïve to elevated arsenic to determine the effects of acclimation on arsenic toxicity and accumulation. Tolerance to arsenic was rapidly (96 h) acquired in killifish that were preexposed. In toxicity tests with arsenic-acclimated killifish, preexposure to 106 μmol As/L resulted in a reduction in toxicity when compared to näive animals. Toxicity in arsenic-acclimated fish also was distinguished by a delayed onset of mortality that manifested in dose-dependent fashion and was significant even for the lower acclimation concentration (0.7 μmol As/L). The increase tolerance acquired following preexposure to 106 μmol As/L for 96 h was associated with lower concentrations of arsenic in all monitored tissues (e.g., gill, liver, kidney) and the whole body when fish were exposed to 240 μmol As/L for an additional 96 h. In accordance with these observations, expression of the multidrug resistance-associated protein (MRP)-2 gene, which is responsible for transporting arsenic conjugated to glutathione out of cells, was increased in the liver of arsenic-acclimated fish.