Low-energy negative-ion electrospray mass spectrometry (ESI-MS) and ESI-MS/MS were used to characterize saturated and unsaturated fatty acids. The carbon number and degree of unsaturation of fatty acids were determined using ESI-MS, and MS/MS was used to localize some double bond positions of mono-and polyunsaturated fatty acids. For compounds with up to two unsaturated bonds, fragmentation was dominated by loss of H,O from the carboxyl moiety and very low-intensity peaks generated from bonds cleaved at carbons a and/or fl to sites of unsaturation. Fragmentation of monounsaturated fatty acids was minimal using this soft method of mass spectrometric analysis, but increased with progressively greater degrees of fatty acid unsaturation. There was extensive hydride migration during ESI-MS/MS of compounds with three or more double bonds. Although this behavior complicated localization of double and triple bonds, the spectra were reproducible. Many peaks could not be definitively assigned to specific product ions, but the spectra of standards and complementary natural products were similar and isobaric compounds could be differentiated. The utility of this technique to examine biological samples was shown by analysis of the fatty acid composition of cod liver oil. Detection limits for negative-ion ESI-MS/MS were at or below 1 pg.