It was previously reported that the glycosylphosphatidylinositol (GPI)-anchored CD55 molecule provides a co-stimulatory signal for T lymphocytes and is constitutively associated with the Src-related kinase p56lck. The present studies were undertaken to clarify the mechanism of action of CD55 in T cells. We describe the failure of cross-linking of CD55 alone to induce both the elevation of the intracellular calcium concentration and the tyrosine phosphorylation of PLC-gamma in CD3+ Jurkat cells. By contrast, it is sufficient to induce the phosphorylation of tyrosine residues on p56lck, the TCR-zeta chain as well as ZAP-70. Surprisingly, the observed TCR-zeta and ZAP-70 tyrosine phosphorylations appear delayed compared to stimulation via CD3. Calcium ionophore A23187 in combination with cross-linked CD55 mAb initially caused an acceleration in the kinetic of these two phosphorylation events, followed by IL-2 secretion. Furthermore, transfection of the cytoplasmic domain of TCR-zeta in CD3- Jurkat cells, using a CD16-zeta chimera, demonstrates that CD55-mediated T-cell activation depends on the expression of this chain of the CD3-TCR complex.