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The S. cerevisisae Rho GTPase Cdc42p localizes around the entire plasma membrane, but is only activated at sites of polarized growth in a cell cycle-dependent manner. This spatial and temporal control ensures that Cdc42p is inactive within the cytoplasm and outside of the polarized cortical domain, and is active at sites of polarized growth. This dissertation addresses spatial control of Cdc42p by examining complex formation with its sole guanine nucleotide dissociation inhibitor (GDI) Rdi1p and its sole guanine nucleotide exchange factor (GEF) Cdc24p. Rdi1p is not essential; however, when overexpressed, it is lethal, suggesting that it does have an important negative regulatory function. To study complex formation and localization, the technique of bimolecular fluorescence complementation (BiFC) was used to study in vivo Rdi1p-Cdc42p complex formation and localization, and how mutations to Rdi1p’s two functional domains, the regulatory arm and the geranylgeranyl binding pocket, affected the complex. Rdi1p and Cdc42p interacted diffusely throughout the cytoplasm o