The CD28 cell surface receptor provides an important costimulatory signal for T cells necessary for their response to Ag. Early events in CD28 signaling include recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase) and activation of the protein tyrosine kinases (PTKs), LCK and EMT. Recruitment and activation of PI3-kinase is known to be dependent upon phosphorylation of tyrosine 173 of the CD28 cytoplasmic tail contained within a YMNM motif. By contrast, little is known of which residues of the CD28 tail, including tyrosines, are required for the activation of PTKs. To address this we studied the ability of truncation mutants and tyrosine to phenylalanine substitution mutants of the CD28 cytoplasmic tail to activate LCK and EMT in Jurkat T leukemia cells. Our results indicate that 1) activation of EMT is partially dependent upon tyrosine 173 of the CD28 tail, although it does not require PI3-kinase activation; 2) activation of LCK is independent of CD28 cytoplasmic tail tyrosine residues; and 3) elements sufficient for the activation of both kinases are contained within the first half of the tail. In addition we studied the CD28 tail as a substrate for both PTKs in in vitro kinase assays. We demonstrate that EMT can phosphorylate all four tyrosines of the CD28 tail, in contrast to LCK, which phosphorylates only tyrosine 173. Together with evidence that in vivo, tyrosines other than tyrosine 173 become phosphorylated following CD28 stimulation, this finding suggests that, like LCK, one function of EMT during CD28 signaling is phosphorylation of the receptor.