Acylphosphatidylglycerol (Acyl-PG), a polar lipid class containing three fatty acyl groups, was isolated from Salmonella bacteria and characterized by tandem quadrupole and quadrupole ion-trap mass spectrometric methods with electrospray ionization. The structural characterization of the acyl-PG with various acyl groups (A-B/C-PG, where A not equal B not equal C) is based on the findings that the carboxylate anions (R(x)CO(2)(-)) arising from sn-2 (R(2)CO(2)(-)) is more abundant than that arising from sn-3' (R(3')CO(2)(-)), which is much more abundant than that arising from sn-1 (R(1)CO(2)(-)). This information provides a simple method for determination of the fatty acyl moieties and their positions in the molecule. The structural identification of the molecule can also be achieved by the findings that the fragment ion reflecting the ketene loss at sn-2 is more prominent than that reflecting the acid loss (i.e., [M - H - R'(2)CH=CO](-) > [M - H - R(2)CO(2)H](-)), while the ion arising from acid loss at sn-1 or sn-3' is, respectively, more abundant than the corresponding ketene loss (i.e., [M - H - R(1)CO(2)H](-) > [M - H - R'(1)CH=CO](-); [M - H - R(3')CO(2)H](-) > [M - H -R'(3')CH=CO](-)). The identity of the acyl moiety at sn-3' can be confirmed by an acyl-glycerophosphate anion observed in the product-ion spectrum obtained with a triple-stage quadrupole (TSQ) instrument, but not in that obtained with an ion-trap mass spectrometer (ITMS). However, the MS(2)-spectrum obtained with an ITMS is featured by the ion series that abundances of [M - H - R'(2)CH=CO - R(3)CO(2)H - 74](-) > [M - H - R'(2)CH=CO - R(1)CO(2)H - 74](-) z.Gt; [M - H - R'(1(or 3'))CH=CO - R(3'(or 1))CO(2)H - 74](-). This information also facilitates structural elucidation of the acyl-PG subclass that contains various acyl substituents. Structural identifications of molecular species having two identical fatty acyl substituents at sn-1, sn-2, or sn-3' or consisting of more than one isomeric structures are also demonstrated. The identities of the minor isomeric species in the molecules can be revealed by the aforementioned structural information arising from the various ion series combined.