In this paper, we present the results of a detailed study using MALDI seamless postsource decay (sPSD) fragment ion analysis of all major glycerophospholipid (GPL) classes, cardiolipin (bisphosphatidylglycerol), and sphingomyelin, respectively. The matrix compound 2,4,6-trihydroxyacetophenon recently introduced for lipid analysis (Stübiger, G.; Belgacem O. Anal. Chem. 2007, 79, 3206-3213) was applied in conjunction with a high-resolution curved field reflectron analyzer allowing detection of the fragment ions without stepping the reflectron voltages (seamless PSD). This instrumental feature helps to define in a fast way the polar headgroups of the different GPL classes and gives information about the constituent fatty acid residues dependent on the type of adduct ion used. Our experiments demonstrate that fragment ions specifying the fatty acid composition of the lipid molecules could only be generated from cationized molecular ions (sodiated or lithiated). Additionally, information about the stereospecificity of the fatty acid residues on the glycerol backbone (sn-1, and -2 position) of particular GPLs could be obtained during sPSD analysis. In the case of phosphatidylcholine, significant fragmentation related to the loss of the acyl groups could only be observed from [M + Li](+) ions. Generally, alkali adduction (sodium and lithium) enhanced fragmentation of most lipid classes, particularly favoring fragment ions associated with the polar headgroups.