The position(s) of carbon-carbon double bonds within lipids can dramatically affect their structure and reactivity and thus has a direct bearing on biological function. Commonly employed mass spectrometric approaches to the characterization of complex lipids however, fail to localize sites of unsaturation within the molecular structure and thus cannot distinguish naturally occurring regioisomers. In a recent communication [Thomas, Mitchell, Blanksby in the Journal of the American Chemical Society, 2006, 128, 58-59] we have presented a new technique for the elucidation of double bond position in glycerophospholipids using ozone-induced fragmentation within the source of a conventional electrospray ionization mass spectrometer. Here we report the on-line analysis, using ozone electrospray mass spectrometry (OzESI-MS), of a broad range of common unsaturated lipids including, acidic and neutral glycerophospholipids, sphingomyelins and triacylglycerols. All lipids analyzed are found to form a pair of chemically induced fragment ions diagnostic of the position of each double bond(s) regardless of the polarity, the number of charges or the adduct ion (e.g., [M-H]-, [M-2H]2-, [M+H]+, [M+Na]+, [M+NH4]+). The ability of OzESI-MS to distinguish lipids that differ only in the position of the double bonds is demonstrated using the glycerophosphocholine standards, GPCho(9Z-18:1/9Z-18:1) and GPCho(6Z-18:1/6Z-18:1). While these regioisomers cannot be differentiated by their conventional tandem mass spectra, the OzESI-MS spectra reveal abundant fragment ions of distinctive mass-to-charge ratio. The approach is found to be sufficiently robust to be used in conjunction with the m/z 184 precursor ion scans commonly employed for the identification of phosphocholine-containing lipids in shotgun lipidomic analyses. This tandem OzESI-MS approach was used - in conjunction with conventional tandem mass spectral analysis - for the structural characterization of an unknown sphingolipid in a crude lipid extract obtained from a human lens. The OzESI-MS data confirm the presence of two regioisomers, namely SM(d18:0/15Z-24:1) and SM(d18:0/17Z-24:1) and suggest the possible presence of a third isomer, SM(d18:0/19Z-24:1), in lower abundance. The data presented herein demonstrate that OzESI-MS is a broadly applicable, on-line approach for structure determination and when used in conjunction with established tandem mass spectrometric methods, can provide near complete structural characterization of a range of important lipid classes. As such, OzESI-MS may provide important new insight into the molecular diversity of naturally occurring lipids.