We investigated the effects of lipids oil thymocyte function. The effects of application of cholesterol or docosahexaenoic acid (DHA). a C22, omega-3 (n-3) polyunsaturated fatty acid (PUFA), on viability and intracellular calcium concentrations of acutely isolated mouse thymocytes were investigated using flow cytometry. Cholesterol (100 mu M) caused significant cell death after 30-60 min whether or not calcium was present in the medium. Cell death was associated with an elevation of intracellular calcium whether or not calcium was present in the extracellular medium. However, the elevation of calcium concentration was not responsible for the cell death since calcium levels in the presence of ionomycin rose higher without significant cell death. DHA had similar actions but was more potent, causing significant cell death and elevation of calcium concentration within 5 min at 1 mu M. In the absence of extracellular calcium 1 mu M DHA caused 100% cell death within 15 min. Linolenic acid, a C18 omega-3 fatty acid also Caused cytotoxicity at low concentrations whether or not albumin was present, but omega-6 or saturated C22 fatty acids were much less effective. These observations demonstrate that thymocyte viability is very sensitive to acute exposure to low concentrations of omega-3 fatty acids. Copyright (c) 2009 John Wiley & Soils, Ltd.