A method for determining the positions of double bonds in unsaturated fatty acids by use of negative electrospray ionisation low-energy tandem mass spectrometry is described. First, a vicinal hydroxylation of the double bonds of mono- and poly-unsaturated fatty acids was performed. Low-energy collision activation dissociation of the deprotonated molecules produced structurally informative ions formed by a-cleavages relative to the hydroxyl groups. Abundant fragment ions that confirmed the positions of all hydroxyl groups, and thus the positions of the double bonds in the native fatty acids, were observed in the spectra of derivatised mono-, di-, and tri-unsaturated fatty acids. Two types of ions were observed, called [alpha'(n)](-) and [alpha(n)](-). The letter n indicates the positions of the hydroxyl groups. The structurally diagnostic ions [alpha'(n)](-) were produced by cleavages distal to the hydroxyl-groups with the charge retained on the carboxylate. [alpha'(n)](-) ions originating from all hydroxyl-groups were observed in the spectra of modified mono-, di-, and tri-unsaturated fatty acids. Initial proton transfer of a hydroxyl proton to the carboxylate with subsequent cleavages proximal to the hydroxyl groups, relative to the carboxylate, resulted in the two structurally diagnostic [alpha(n)](-) ions. In hydroxylated fatty acids having two or more double bonds in their native structure, [alpha(n)](-) ions originating only from the two final hydroxyl-groups were observed. The formation of all ions of [alpha'(n)](-) and [alpha(n)](-) type can be rationalised by a six-membered transition state. Hydroxylated deprotonated tetra-, penta-, and hexa-unsaturated fatty acids also produced [alpha'(n)](-) ions indicating the positions of most of the hydroxyl-groups, whereas the [alpha(n)](-) ions were observed as described above. The method described offers a simple approach to the determination of the positions of double bonds in unsaturated fatty acids, and is an alternative to utilising di-lithiated fatty acids. The method should also be applicable when fatty acids are constituents of more complex molecules such as phospholipids.