A regulatory protein for a liver GTP-binding protein (G protein) with a molecular weight value of 24,000 (24K G), which we have recently purified, was purified to near-homogeneity from rat liver cytosol and characterized. This regulatory protein, designated here as GDP dissociation inhibitor for 24K G (24K G GDI), inhibited the dissociation of GDP from and the subsequent binding of GTP to 24K G. 24K G GDI was inactive for other ras p21/ras p21-like small G proteins including c-Ha-ras p21, rhoB p20, smg p21B, and smg p25A. 24K G was, however, recognized by bovine brain smg p25A GDI which regulated the GDP/GTP exchange reaction of smg p25A. By analyses of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), immunoblotting with anti-smg p25A GDI antibody, two-dimensional PAGE, and C4 column chromatography, 24K G GDI showed physical properties very similar to those of smg p25A GDI. The peptide map and the partial amino acid sequences of 24K G GDI were not identical with those of smg p25A GDI. Among the 83 residues, 2 amino acids were different between rat liver 24K G GDI and bovine brain smg p25A GDI. These results indicate that there is a specific regulatory protein for 24K G, 24K G GDI, in rat liver cytosol and that 24K G GDI has close similarity to smg p25A GDI.