The Raf kinase inhibitor protein (RKIP) increasingly evolves as an important regulator of intracellular signaling networks and thus participates in diverse physiological functions ranging from growth and differentiation processes to muscle contraction. Several molecular events contribute to the ability of RKIP to tightly coordinate kinase signaling. The elucidation of the molecular mechanisms leading to substrate specificity of RKIP and substrate binding efficacy is of great interest for a better understanding of the overall role of RKIP in the organism but also for the design of specific and potent kinase inhibitors. In this work, we will review mechanistic details of the regulation of RKIP as inhibitor of Raf-1 and G protein-coupled receptor kinase 2 (GRK2) that enable RKIP to coordinate the cell's balance between inhibition and potentiation of mitogenic ERK1/2 signaling--a prominent example of RKIP's function as a regulator of intracellular signaling.