Rho GTPases play a crucial role in regulating various cellular processes, including actin cytoskeleton dynamics, cell polarity, gene expression, and cell cycle progression. Their activity is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the exchange of GDP for GTP, and guanine nucleotide dissociation inhibitors (GDIs), which control the cycling between membrane and cytosol. The DH domain of GEFs is essential for nucleotide exchange, while the PH domain helps target GEFs to specific locations. GEFs are regulated through intramolecular inhibitory sequences, protein-protein interactions, and changes in subcellular localization. In higher eukaryotes, the number of GEFs exceeds that of their GTPase substrates, suggesting that multiple GEFs may activate the same GTPase. This redundancy allows GEFs to influence the selection of downstream pathways and subcellular localization. Examples of GEFs in action include DRhoGEF2 in gastrulation, Tiam1 in neuronal morphogenesis, Trio and Ephexin in axon guidance, and Vav in immune responses. Genetic alterations in GEFs are associated with developmental disorders, neurodegenerative diseases, and cancer, highlighting their importance in human biology.Rho GTPases play a crucial role in regulating various cellular processes, including actin cytoskeleton dynamics, cell polarity, gene expression, and cell cycle progression. Their activity is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the exchange of GDP for GTP, and guanine nucleotide dissociation inhibitors (GDIs), which control the cycling between membrane and cytosol. The DH domain of GEFs is essential for nucleotide exchange, while the PH domain helps target GEFs to specific locations. GEFs are regulated through intramolecular inhibitory sequences, protein-protein interactions, and changes in subcellular localization. In higher eukaryotes, the number of GEFs exceeds that of their GTPase substrates, suggesting that multiple GEFs may activate the same GTPase. This redundancy allows GEFs to influence the selection of downstream pathways and subcellular localization. Examples of GEFs in action include DRhoGEF2 in gastrulation, Tiam1 in neuronal morphogenesis, Trio and Ephexin in axon guidance, and Vav in immune responses. Genetic alterations in GEFs are associated with developmental disorders, neurodegenerative diseases, and cancer, highlighting their importance in human biology.