Ras-related small GTP-binding proteins (SMGs) exist in a cytosolic and a membrane-bound pool. The mechanism regulating the intracellular distribution of SMGs remains to be elucidated. We have, therefore, investigated the properties of SMGs expressed in cells of the insulin-secreting lines RINm5F and HIT-T15. Phase-partitioning analysis revealed that smg25A/rab3A as well as all the SMGs in the 23-27 kDa range, labeled by radioactive GTP after blotting, were hydrophobic, regardless of their subcellular distribution. In contrast, the cytosolic forms of ADP ribosylation factor, rho, and CDC42 were hydrophilic. The cytosolic pool of the 23-27-kDa group, including smg25A/rab3A, sedimented in a sucrose density gradient as complexes with an apparent M(r) of about 80,000, whereas rho and CDC42 were recovered in 45-kDa complexes. ARF, however, was uncomplexed (M(r) close to 20,000). The 80-kDa aggregates were likely to be formed by 1:1 complexes with the regulatory protein smg25/GDP dissociation inhibitor (smg25/GDI). In fact, pure smg25/GDI by sucrose gradient exhibited a molecular mass of 55 kDa, but cosedimented with the 80-kDa complexes in cytosolic extracts of insulin-secreting cells. Moreover, purified smg25/GDI was able to extract the SMGs of the 23-27-kDa group from the membranes. Similarly, in cytosolic extracts, rho/GDI cosedimented with the 45-kDa aggregates. Blocking the synthesis of isoprenoid groups with lovastatin resulted in the appearance in the cytosol of SMGs that were hydrophilic. These SMGs were found to sediment with an apparent M(r) close to 25,000 and to be unable to form complexes with smg25/GDI. Lovastatin treatment also caused the accumulation of the noncomplexed form of CDC42 but not of rho proteins. We propose that 1) except for ARF, all the SMGs detected in the cytosol of insulin-secreting cells are associated in 1:1 complexes with their regulatory proteins; 2) the different SMGs can be subdivided into functional groups according to the regulatory protein bound to them; 3) the formation of the 80-kDa complexes with smg25/GDI and of the CDC42 complexes with rho/GDI necessitate the correct carboxyl-terminal post-translational modification of the SMGs.