One of the earliest events following T cell receptor (TCR) triggering is the activation of the protein kinase Lck and induction of tyrosine phosphorylation of zeta, the major signal transduction subunit of the T cell receptor complex. Here we report the generation and characterization of a monoclonal antibody specific for human phosphozeta. The antibody was produced by immunizing mice with a truncated recombinant form of human zeta together with the Lck enzyme. The C415.9A antibody recognizes recombinant as well as cellular phosphozeta but is unreactive with unphosphorylated zeta or other tyrosine phosphorylated proteins. Using this antibody, we have demonstrated aberrant TCR-zeta tyrosine phosphorylation in Jurkat T cell transduction mutants. Therefore, this antibody can be used to elucidate T cell signal transduction mechanisms by analyzing and monitoring tyrosine phosphorylation of zeta in vitro and in vivo directly. Furthermore, this antibody could find application in the analysis of abnormal T cell signaling in autoimmune disease, cancer, and immunodeficiency disorders.