Protein L12, together with the P0/P1/P2 protein complex, forms the protein moiety of the GTPase domain in the eukaryotic ribosome. In Saccharomyces cerevisiae protein L12 is encoded by a duplicated gene, rpL12A and rpL12B. Inactivation of both copies has been performed and confirmed by Southern and Western analyses. The resulting strains are viable but grow very slowly. Growth rate is recovered upon transformation with an intact copy of the L12 gene. Ribosomes from the disrupted strain lack protein L12 but are able to carry out translation in vitro at about one fourth of the control rate. The L12-deficient ribosomes have also a defective stalk containing standard amounts of the 12-kDa acidic proteins P1beta and P2alpha, but proteins P1alpha and P2beta are drastically reduced. Moreover, the affinity of P0 is reduced in the defective ribosomes. Footprinting of the 26 S rRNA GTPase domain indicates that protein L12 protects in different extent residues G1235, G1242, A1262, A1270, and A1272 from chemical modification. The results in this report indicate that protein L12 is not essential for cell viability but has a relevant role in the structure and stability of the eukaryotic ribosomal stalk.