We previously demonstrated that altered peptide ligands (APL) can partially activate T cells, resulting in multiple distinct functional phenotypes, including the induction of anergy. Such APL stimulate a unique pattern of T cell receptor (TCR) phospho-zeta species, and lack associated ZAP-70 kinase activity. While these data suggested that selective signaling pathways downstream of the TCR/CD3 molecules are activated upon APL stimulation, they did not directly demonstrate this. Thus, we pursued intracellular signaling events successfully stimulated by APL. Because our previous studies showed that cyclosporin A (CsA) completely inhibited anergy induction, we assessed whether TCR ligation by APL cause a rise in cytosolic calcium (Ca+2). Our results show that these ligands can induce Ca+2 transients, in contrast to data generated using analogue peptides in other antigen systems. These opposing results may reflect differences in the intracellular signaling pathways utilized by different APL, or may be due to the exquisite sensitivity of the assay used here. Importantly, the APL-stimulated Ca+2 induction is both initiated and sustained at lower levels than that stimulated by a strong agonist signal, but resembles that stimulated by a weaker agonist stimulus. Alone, the less than optimal Ca+2 induction does not cause anergy, because ionomycin treatment together with the APL does not result in a proliferative signal. Instead, we propose that a combination of this and other signaling pathways induces T cell anergy. Overall, these data support the concept of differential signaling in T cells, as a direct consequence of the phosphotyrosine status of the TCR/CD3 molecules.