Tec family non-receptor tyrosine kinases (Itk, Btk, Tec, Rlk and Bmx) are characterized by the presence of an autophosphorylation site within the non-catalytic Src homology 3 (SH3) domain. The full length Itk mutant containing a phenylalanine in place of the autophosphorylated tyrosine has been previously studied in Itk deficient primary T cells. These studies revealed that the nonphosphorylated enzyme only partially restores Itk mediated signaling. In spite of these insights, the precise role of the Tec kinase autophosphorylation site remains unclear and the mechanism of the autophosphorylation reaction within the Tec kinases is not known. Here we show both in vitro and in vivo that Itk autophosphorylation on Y180 within the SH3 domain occurs exclusively via an intramolecular, in cis mechanism. Using an in vitro kinase assay we also show that mutation of the Itk autophosphorylation site Y180 to Phe decreases kinase activity of the full-length enzyme by increasing Km for a peptide substrate. Moreover, mutation of Y180 to Glu, a residue chosen to mimic the phosphorylated tyrosine, alters the ligand binding capability of the Itk SH3 domain in a ligand dependent fashion. NMR chemical shift mapping gives residue-specific structural insight into the effect of the Y180E mutation on ligand binding. These data provide a molecular level context with which to interpret in vivo functional data and allow development of a structural model for Itk autophosphorylation.