Talin, which is composed of head (THD) and rod domains, plays an important role in cell adhesion events in diverse species including most metazoans and Dictyostelium discoideum. Talin is abundant in the cytosol; however, it mediates adhesion by associating with integrins in the plasma membrane where it forms a primary link between integrins and the actin cytoskeleton. Cells modulate the partitioning of talin between the plasma membrane and the cytosol to control cell adhesion. Here, we combine nuclear magnetic resonance spectroscopy (NMR) with subcellular fractionation to characterize two distinct THD-rod domain interactions that control the interaction of talin with the actin cytoskeleton or its localization to the plasma membrane. An interaction between a discrete vinculin-binding region of the rod (VBS1/2a; Tln1(482-787)), and the THD restrains talin from interacting with the plasma membrane. Furthermore, we show that vinculin binding to VBS1/2a results in talin recruitment to the plasma membrane. Thus, we have structurally defined specific inter-domain interactions between THD and the talin rod domain that regulate the subcellular localization of talin.