Cellular mechanisms for controlling membrane trafficking appear to involve small GTP-binding proteins such as the Rab proteins. Rab function is regulated by GDP dissociation inhibitor (GDI), which releases Rab proteins from membranes and inhibits GDP dissociation. Here we report the isolation of a full-length cDNA encoding a novel GDI isoform of 445 amino acids (GDI-2) with a deduced molecular weight of 50,649 from mouse skeletal muscle. Full-length and partial cDNA clones encoding a previously reported GDI protein (GDI-1) were also isolated from cDNA libraries prepared from rat brain and mouse skeletal muscle, respectively. The degree of deduced amino acid sequence identity between mouse GDI-2 and our mouse GDI-1 cDNA clone is 86%. Northern (RNA blot) analysis revealed that in human tissues, both GDI-1 and GDI-2 transcripts were abundant in brain, skeletal muscle, and pancreas but were weakly expressed in heart and liver. GDI-1 mRNA was expressed in kidney, whereas GDI-2 was almost absent, while in lung the relative amounts of these mRNA species were reversed. Specific antibodies against mouse GDI-1 and GDI-2 based on unique peptide sequences in the proteins were raised. Differentiation of 3T3-L1 fibroblasts into highly insulin-responsive adipocytes was accompanied by large increases in both mRNA and protein levels of GDI-1 and GDI-2. GDI-1 and GDI-2 expressed as glutathione S-transferase fusion proteins were both able to solubilize the membrane-bound forms of Rab4 and Rab5 in a GDP/GTP-dependent manner. Taken together, these data demonstrate that the protein products of at least two genes regulate the membrane dynamics of Rab proteins in mice.