We have recently shown that altered peptide ligands influence differentiation of CD4+ T cells into Th1 and Th2 subsets. In the present study, we have examined the biochemical signals in naive CD4+ T cells after priming with altered peptide ligand (APL) that correlate with differences in cytokine expression. Although we observed zeta-chain phosphorylation in APL-stimulated cells, other signaling events such as ZAP70 and Lnk phosphorylation are not initiated. This altered pattern observed in the early phosphorylation events correlates with a distinct Ca2+ mobilization pattern that characterizes APL-stimulated cells. By changing the calcium signaling environment during T cell priming, we present data indicating that qualitative differences in calcium mobilization are associated with differentiation of naive CD4+ T cells into Th1- and Th2-like effector subsets.