Ligation of the TCR-CD3 complex in T lymphocytes released inositol trisphosphate (IP3) and its breakdown products IP2 and IP1 into the cytoplasm as a result of the hydrolysis of membrane phosphatidyl inositol 4,5-bisphosphate (PtdIns[4,5]-P2). This was associated with the incorporation of free phosphoinositides into the lipid membrane in the form of PtdIns, PtdIns(4)-P, and PtdIns(4,5)-P2. Activation of T lymphocytes with monocyte chemoattractant protein-1, a chemokine that only transiently activates phospholipase C (PLC), led to the release of IP3 and its metabolites into the cytoplasm without resynthesis of membrane phosphoinositides; thus, breakdown and resynthesis of phosphoinositides was found to be specific to TCR signals. Addition of the CD28 signal to the TCR-CD3 signal led to dramatic augmentation of phosphoinositide incorporation in T-lymphocyte membranes, and prolongation of the intracellular Ca2+ (Cai) signal. Pharmacologic inhibition of Src protein tyrosine kinase (PTK) and PLC?1 obliterated the co-stimulatory effects on Cai levels.