It is now well-recognized that the spatial localization of proteins at the subcellular level is essential in regulating cell signaling. On the other hand, research efforts aimed at developing synthetic modulators of intracellular signaling processes have usually focused on the affinity and specificity of ligands to their target proteins, neglecting the localization of the molecules in cells. Here we show that it is feasible to rapidly and efficiently activate an endogenous signaling pathway by placing a synthetic ligand at a specific location within a cell. The agonist activity of the ligand is very weak when it is freely diffusing in the cytosol. This study highlights the importance of controlling the subcellular locations of ligand molecules in the design of new synthetic modulators (not only activators but also inhibitors) of endogenous biological events.