The gamma subunit of the human T lymphocyte T3 antigen is rapidly phosphorylated on serine residues in vivo during the initiation of T cell activation by a polyclonal mitogen (Phaseolus vulgaris phytohemagglutinin), an activator of protein kinase C (phorbol 12,13-dibutyrate), and an elevator of intracellular calcium (ionomycin). The sites of phosphorylation were identified by comparing tryptic peptide analyses of T3 gamma chains labeled in vivo with various synthetic peptides, corresponding to portions of the cytoplasmic domain of the gamma chain that had been labeled in vitro using purified protein kinase C. Two sites, serines 123 and 126, were phosphorylated in response to ionomycin, whereas a single site, serine 126, was phosphorylated when T lymphocytes were stimulated by P. vulgaris phytohemagglutinin or when protein kinase C was directly activated by phorbol 12,13-dibutyrate. Immune activation of T cells via the protein kinase C pathway thus induces phosphorylation of a single site on the T3 gamma chain, namely serine 126.