During CsCl isopycnic centrifugation at 20 mM Mg++, Escherichia coli 30S ribosomal subunits specifically lose proteins S1, S2, S3, S5, S9, S10, and S14. The resultant 30S core is unable to stimulate the GTPase activity of EF-G in the presence of 50S subunits. Activity could be restored to a small extent by adding back S2, S5, or S9. However, when S5 and S9 were added together, they cooperatively produced 30S particles 1.5 times more active than the original native 30S subunits. The small amount of activity restored by S2 was simply additive to that restored by S5 or S9. None of the other split proteins showed any restoring capability. Ability of the various protein-deficient 30S particles to couple with 50S subunits corresponded closely to their activity in the EF-G GTPase reaction. It is concluded that S5 and S9 together enable the 30S subunit to participate in the formation of a GTPase-active 30S-50S-EF-G complex.