Arachidonic acid is oxidized to regioisomeric 5(S)-, 12(S)- and 15(S)-hydroxyeicosatetraenoic acids by the corresponding 5-, 12- and 15-lipoxygenases. These hydroxylated fatty acids can then be incorporated into cellular phospholipids. Negative liquid secondary ion tandem mass spectrometry using a high-energy collision regime in a tandem four-sector mass spectrometer was used to characterize regioisomeric hydroxyeicosatetraenoic acids and the corresponding hydroxyeicosatetraenoic phosphatidylcholine species. Collision-induced dissociation (CID) of the [M-H]- negative ion at m/z 319 from the hydroxyeicosatetraenoic acids regioisomers produced some similar product ions, such as m/z 301 [M-H-H2O]- and m/z 257 [M-H-(H2O + CO2)]-. In addition, product ions characteristic of the particular hydroxyeicosatetraenoic acid were formed from alpha-cleavages adjacent to the hydroxyl moieties. Negative liquid secondary ion mass spectrometry of purified hydroxyeicosatetraenoate phosphatidylcholine species gave an ion at m/z 810 [M-CH3]-. CID of the m/z 810 ion gave product ions at m/z 283 and m/z 319, corresponding to stearate at the sn-1 position and hydroxyeicosatetraenoate at the sn-2 position, respectively. From CID of the negative ion at m/z 319 and examination of the product ion spectra, the hydroxyeicosatetraenoate regioisomer present in the phosphatidylcholine could be identified.