To determine whether altered peptide ligands (APL) affect calcium signaling events, we investigated changes in intracellular calcium concentration ([Ca2+]i) in human T cell clone stimulated with either the fully agonistic peptide M12p54-68, the partially agonistic analogue E63V or the simple antagonistic analogue E58M. Both E63V and E58M stimulated a Ca2+ response in approximately 40% of T cells, whereas M12p54-68 did so in approximately 70% of T cells. The most predominant pattern of a Ca2+ increase induced by M12p54-68 was a small sinusoidal peak followed by a sustained high response. The most frequent pattern of calcium response induced by E63V was a continuous high response without a preceding sinusoidal peak, whereas that induced by E58M was large with frequent oscillations. Genistein, an inhibitor of the protein tyrosine kinases (PTK), markedly inhibited the wild-type peptide-induced increase in [Ca2+]i, whereas it marginally inhibited the response induced by E63V or E58M. In contrast, GF109203X, a protein kinase C (PKC)-specific inhibitor, markedly inhibited the E63V- or E58M-induced Ca2+ response, whereas it marginally affected the wild peptide-induced Ca2+ response. Furthermore, in nominal Ca2+-free medium, the E58M-induced Ca2+ response was almost completely blocked, while the M12p54-68- or E63V-induced responses were only partially inhibited. Our results suggest that the Ca2+ response induced by the fully agonistic peptide depends on activation of the genistein-sensitive signaling pathway, including PTK, whereas the Ca2+ response to a simple antagonistic APL completely depends on extracellular Ca2+ and activation of the GF109203X-sensitive signaling pathway, including PKC. These differences in the CA2+i response in recognition of different APL may parallel the unique T cell activation patterns induced by APL in human T cells.