The development of a new mass spectrometric lipid profiling methodology permits the identification of cellular phosphatidylinositol monophosphate/phosphatidylinositol bisphosphate/phosphatidylinositol trisphosphate (PIP/PIP2/PIP3) species that includes the fatty acyl composition. Using electrospray ionization mass spectrometry, we were able to resolve and identify 28 PIP and PIP2 compounds as well as 8 PIP3 compounds from RAW 264.7 or primary murine macrophage cell extracts. Analysis of PIP profiles after agonist stimulation of cells revealed the generation of differential PIP3 species and permitted us to propose a novel means for regulation and specificity in signaling through PIP3. This is the first reported identification of intact, cellular PIP3 by mass spectral analysis. The ability to analyze the fatty acyl chain composition of signaling lipids initiates new venues for investigation of the processes by which specific polyphosphoinositide species mediate.