Oxidative stress in T cells induces signaling events similar to those initiated by T cell antigen receptor engagement, including tyrosine phosphorylation and activation of the critical protein-tyrosine kinase ZAP-70. Distal signaling events such as the activation of mitogen-activated protein kinases and downstream transcription factors are also initiated by oxidative stimuli. In this study P116, a ZAP-70-negative Jurkat T cell line, was used to investigate the role of ZAP-70 in mediating activation of Erk in response to H2O2. Consistent with the hypothesis that ZAP-70 is required for activation of Erk in response to an oxidative stimulus, Erk1 and Erk2 could be rapidly activated in Jurkat cells but not in P116 cells upon addition of H2O2. P116 cells became competent for H2O2-induced Erk activation upon stable transfection with wild-type ZAP-70. An in vivo ZAP-70 substrate, SLP-76, implicated in Erk activation, also became rapidly tyrosine-phosphorylated in Jurkat cells, but not in P116 cells, upon treatment with H2O2. Surprisingly, although ZAP-70 was required for H2O2-mediated Erk activation, Erk activation in response to T cell antigen receptor engagement did not require ZAP-70. In addition to demonstrating a requirement for ZAP-70 in H2O2-stimulated Erk activation, these results provide the first evidence for the existence of a ZAP-70-independent pathway for Erk activation in T cells.