A combination of metabolic labeling and chemical or enzymatic modification was employed to isolate and biochemically characterize a set of glycosyl-phosphatidylinositol (gly-PI) molecules synthesized by T lymphocytes. Gly-PI displayed unique patterns of synthesis following mitogen activation relative to the phosphoinositides and major structural lipids. The increase with time in gly-PI was paralleled by the appearance of insulin receptors. Gly-PI molecules were sensitive to hydrolysis by a PI-specific phospholipase C and were rapidly (15 sec) degraded in response to insulin binding. The product of this hydrolysis is believed to be a novel inositol phosphate-glycan (IP-gly) that was shown to inhibit the activity of a cAMP-dependent protein kinase. These results demonstrate that T cells contain a structurally related set of gly-PI molecules, at least one of which is sensitive to insulin and may function as a second messenger of hormone action.