Ionization and prompt fragmentation patterns of triacylglycerols, phospholipids (PLs) and galactolipids were investigated using matrix-assisted laser desorption/ionization (MALDI). Positive ions of non-nitrogen-containing lipids appeared only in the sodiated form, while nitrogen-containing lipids were detected as both sodiated and protonated adducts. Lipids containing acidic hydroxyls were detected as multiple sodium adducts or deprotonated ions in the positive and negative modes, respectively, with the exception of phosphatidylcholines. The positive MALDI spectra of triacylglycerols contained prompt fragments equivalent to the loss of RCOO− from the neutral molecules. Prompt fragment ions [PL–polar head]+ were observed in the positive MALDI spectra of all phospholipids except phosphatidylcholines. The phosphatidylcholines produced only a minor positive fragment corresponding to the head group itself (m/z 184). Galactolipids did not undergo prompt fragmentation. Post-source decay (PSD) was used to examine the source of prompt fragments. PSD fragment patterns indicated that the lipid prompt fragment ions did not originate from the observed molecular ions (sodiated or protonated), and suggested that the prompt fragmentation followed the formation of highly unstable, probably protonated, precursor ions. Pathways leading to the formation of prompt fragment ions are proposed. Copyright © 2002 John Wiley & Sons, Ltd.