A single-cell assay was developed to measure the activation of phosphoinositide 3-kinase (PI3K) using microanalytical chemical separations and a fluorescently labeled lipid substrate. Phosphatidyl-inositol 4,5 bisphosphate labeled on its acyl chain with Bodipy fluorescein (Bodipy Fl PIP₂) was utilized as a substrate for both in vitro and cell-based assays. Detection limits for the substrate and product of the PI3K reaction were 10 to 20 zeptomol. In vitro assays with PI3K with and without pharmacologic inhibitors demonstrated that Bodipy Fl PIP₂ was converted to phosphatidyl-inositol 3,4,5 trisphosphate (Bodipy Fl PIP₃). Bodipy Fl PIP₃ could be back converted to Bodipy Fl PIP₂ by the phosphatase PTEN. When Bodipy Fl PIP₂ was added to a cell lysate, 1.4 fmol of the Bodipy Fl PIP₃ were produced per ng of protein in the cytoplasmic extract in 10 min. Addition of Bodipy Fl PIP₃ to a cell lysate yielded 3 fmol of Bodipy Fl PIP₂ per ng of protein in 8 min. Both Bodipy Fl PIP₂ and Bodipy Fl PIP₃ were measureable in single cells and the two species could be inter-converted. Under the appropriate conditions, a fluorescent diacylglycerol was also detected in single cells. When the FcεR1 receptor on the cells loaded with the fluorescent lipid was cross-linked, the amount of Bodipy Fl PIP₃ generated per cell increased 4-fold over that of unstimulated cells. This production of Bodipy Fl PIP₃ was blocked by wortmannin. Chemical cytometry utilizing the fluorescent lipids will be of value in understanding lipid metabolism at the single-cell level.