The remarkable sensitivity of electrospray ionization was exploited to achieve great increases in the sensitivity of tandem mass spectrometric analyses of phospholipids derived from both synthetic and biologic sources. Herein, we demonstrate that (1) product-ion spectra after electrospray ionization can be obtained easily by utilizing ≤ 5 pmol of phospholipid with a mass-selected window of less than 2 mass units, (2) the low energy inherent in the electrospray ionization method facilitates analysis of labile molecular ions that are not easily detected with the relatively high energy employed during fast-atom bombardment desorption, and (3) collision-induced dissociation of precursor ions generated from electrospray ionization often resulted in novel product-ion patterns. Collectively, these results underscore the utility of electrospray ionization tandem mass spectroscopy for the structural determination of diminutive amounts of phospholipids.