Initiation of T-cell antigen receptor (TCR) signaling is dependent upon the activity of protein tyrosine kinases. The Src family kinase Lck is required for the initial events in TCR signaling, such as the phosphorylation of the TCR complex and the activation of ZAP-70, but little is known of its role in downstream signaling. Expression of a mutated form of Lck lacking SH3 domain function (LckW97A) in the Lck-deficient T-cell line JCaM1 revealed a requirement for Lck beyond the initiation of TCR signaling. In cells expressing LckW97A, stimulation of the TCR failed to activate the mitogen-activated protein kinase (MAPK) pathway, despite normal TCR zeta chain phosphorylation, ZAP-70 recruitment, and ZAP-70 activation. Activation of extracellular signal-regulated kinase (ERK) and MAPK kinase (MEK), as well as the induction of CD69 expression, was greatly impaired in JCaM1/LckW97A cells. In contrast, the phosphorylation of phospholipase Cgamma1 (PLCgamma1) and corresponding elevations in intracellular calcium concentration ([Ca2+]i) were intact. Thus, cells expressing LckW97A exhibit a selective defect in the activation of the MAPK pathway. These results demonstrate that Lck has a role in the activation of signaling pathways beyond the initiation of TCR signaling and suggest that the MAPK pathway may be selectively controlled by regulating the function of Lck.