The facile structural determination of lysophospholipid regioisomers represents a long-standing problem in phospholipid chemistry. Herein we report that positive-ion electrospray ionization (ESI) tandem mass spectrometry of sodiated lysophospholipid regioisomers results in the presence of multiple diagnostic pairs of product ions which allows the rapid and direct discrimination between sn-1-acyl- and sn-2-acyllysophospholipid regioisomers. For example, after ESI in the positive-ion mode and subsequent collision-induced dissociation, over a 30-fold difference in the peak intensity ratio of product ions at mit 104 and 147 was manifest with sodiated sn-1-acyllysophosphatidylcholine in comparison to sn-2-acyllysophosphatidylcholine. The observed differences in precursor ion dissociation rates reflect both the kinetically favored formation of a 5-membered phosphodiester (compared to the corresponding 6-membered phosphodiester) and the accelerated rate of an activated vs a nonactivated rearrangement. The structure of individual molecular species and classes of lysophospholipids was substantiated after ESI in the negative-ion mode by tandem mass spectrometry which facilitated identification of lysophospholipid aliphatic constituents and polar head groups. Thus, by exploiting the remarkable sensitivity of electrospray ionization in conjunction with the combined utilization of tandem mass spectrometry in both the positive- and negative-ion modes, structural determination of the specific regioisomer, individual molecular species, and class of lysophospholipids is possible from picomole amounts of material.