Ras-like GTPases contain a structurally conserved GTP-binding domain. An important element of the GTP-binding domain is the phosphate-binding loop, which contains two Gly residues (Gly(12) and Gly(13)) in Ras. Because the two Gly residues are crucial for normal Ras function, it is intriguing that they are not conserved in other Ras-like GTPases, including the Rab GTPases; for example, the equivalent residues in Rab5 are Ser(29) and Ala(30). The present study builds on earlier biochemical characterizations of the Rab5 mutants containing substitutions at Ala(30) and provides a comprehensive analysis of the structure-function relationship of the Rab5 phosphate-binding loop. We have generated 19 new mutants containing amino acid substitutions at Ser(29) and determined whether these Ser(29) mutants, as well as the Ala(30) mutants, remain able to stimulate the endocytosis of horseradish peroxidase in baby hamster kidney cells. A total of 11 mutants lose the activity of stimulating endocytosis. Of these 11 mutants, 9 are defective in membrane association. In contrast, 27 mutants remain able to stimulate endocytosis. Five of them induce a novel cellular phenotype: cell rounding and detachment from culture dishes. They also induce super-large early endosomes such as the constitutively activated Rab5:Q79L mutant. Biochemical results suggest that the constitutive activation of Rab5 requires an increased nucleotide exchange rate and/or decreased GTPase activity. This study establishes functional significance for the phosphate-binding loop of Rab5 and shows that mutations in this region lead to either a loss-of-function or a gain-of-function phenotype, indicating a structure-function relationship distinct from that of Ras.