Every cell contains many families of protein kinases, and may express several structurally related yet genetically distinct kinases of each family. The activity of the serine/threonine protein kinase C (PKC) enzymes has long been implicated in T-cell activation, but it is not known which members of the PKC family regulate the T-cell response to foreign antigens. The activation of T cells by antigen-presenting cells (APCs) is spatially restricted to their site of contact, where receptors on the T cells engage their counter-receptors on the APCs. We used this localized engagement to identify, at the single-cell level, intracellular proteins involved in the activation process. By digital immunofluorescence microscopy, we localized six isoforms of PKC in antigen-specific T-cell clones activated by APCs. Surprisingly, only PKC-theta translocated to the site of cell contact. Accordingly, in vitro kinase activity assays of PKC immunoprecipitates from the conjugates of T cells and APCs showed a selective increase in the activity of PKC-theta, indicating that the translocated enzyme is active. Several modes of partial T-cell activation that failed to cause PKC-theta translocation also failed to cause T-cell proliferation, further suggesting the involvement of PKC-theta in T-cell activation.