The immunological synapse (IS) formed between a T cell and its cognate antigen-presenting cell (APC) enables the directional secretion of cytolytic and inflammatory molecules. Synaptic architecture is established in part by a two-step cascade of novel protein kinase C (nPKC) isozymes. PKCε and PKCη arrive at the IS first, and occupy the entire synaptic membrane. Then, PKCθ accumulates in a smaller zone at the center of the contact. We investigated the molecular basis for this differential recruitment behavior using chimeric nPKC constructs and total internal reflection fluorescence microscopy. Our studies revealed that the V3 linker just N-terminal to the kinase domain plays a crucial role in specifying nPKC localization. Substitution of this linker switched the scope and the kinetics of PKCθ accumulation to that of PKCε and PKCη, and vice versa. Although the V3 was necessary for synaptic compartmentalization, it was not sufficient, as the tandem C1 domains were also required to mediate membrane association. Together, these results suggest a model whereby the V3 linker controls nPKC sub-compartmentalization after initial C1 domain-mediated accumulation at the IS.