The class of long chain polyunsaturated fatty acids known as omega-3 are believed to be involved in prevention of a number of human afflictions. The mode of action for two of the most common omega-3 fatty acids, linolenic 18:3 delta 9,12,15 and docosahexaenoic 22:6 delta 4,7,10,13,16,19 (DHA), is not known. One suggestion is that they may be incorporated into membranes and there provide some specific function. Here we compare the effects of DHA and its metabolic precursor linolenic acid on the membrane properties of fluidity, fusion and permeability. The fatty acids were investigated as both free fatty acids and mixed chain 18:0, 18:3 and 18:0, 22:6 phosphatidylcholines (PCs). Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and a series of anthracene stearic acid probes indicates 20 mol% incorporation of either fatty acid into dipalmitoylphosphatidylcholine bilayers broadens and depresses the temperature of the phase transition, but has almost no effect on fluidity in the liquid crystalline state. Similar fluidity was also observed in the liquid crystalline bilayers of the mixed chain PCs using the same set of fluorescent fatty acid probes. In contrast, DHA as a free fatty acid or as part of a mixed chain PC, causes a much greater enhancement than linolenic acid of the rates of fusion and permeability as monitored by fluorescence resonance energy transfer and aqueous compartment mixing (fusion) and by lipid vesicle swelling in isotonic erythritol, (permeability). These experiments establish a clear distinction between the effects of linolenic acid and DHA in membranes.