T cell receptor (TCR) signal transduction is mediated by the immunoreceptor tyrosine-based activation motifs (ITAM). The ten ITAM in the TCR complex are distributed in two distinct signaling modules termed TCR ζζ and CD3 γϵ/δϵ. To delineate the specific role of the ζ ITAM in T cell development and TCR signal transmission, we compared the properties of T cells from different TCR ζ-transgenic lines wherein tyrosine-to-phenylalanine substitutions had been introduced in the ζ subunit. These lines lack selected phosphorylated forms of TCR ζ including just p23, both p21 and p23, or all phospho-ζ derivatives. We report herein that the efficiency of positive selection in HY TCR-transgenic female mice was directly related to the number of ζ ITAM in the TCR. In contrast, TCR-mediated signal transmission and T cell proliferative responses following agonist peptide stimulation were similar and independent of the ζ ITAM. Only the duration of MAPK activation was affected by multiple ζ ITAM substitutions. These results strongly suggest that the ITAM in the CD3 γϵ/δϵ module can provide normal TCR signal transmission, with ζ ITAM providing a secondary function facilitating MAPK activation and positive selection.