The secretion of Yops via the Yersinia type III secretion system (T3SS) is controlled, in part, by a cytoplasmic YopN/TyeA complex. This complex is required to prevent Yop secretion in the presence of extracellular calcium and prior to contact between the bacterium and a eukaryotic cell. In this study we utilized site-directed mutagenesis to analyze the role of specific TyeA regions and residues in the regulation of Yop secretion. We identified two spatially distinct, surface-exposed regions of the TyeA molecule that were required to regulate Yop secretion. One region, identified by residues M51, F55 and P56, was required for TyeA to interact with YopN. A second region, identified by residues R19, W20 and D25 was not involved in the interaction of TyeA with YopN, but may be required for the YopN/TyeA complex to interact with the T3S apparatus in a manner that blocks Yop secretion.