Yeast ribosomal protein L41 is dispensable in the yeast. Its absence had no effect on polyphenylalanine synthesis activity, and a limited effect on growth, translational accuracy, or the resistance toward the antibiotic paromomycin. Removal of L41 did not affect the 60:40 S ratio, but it reduced the amount of 80 S, suggesting that L41 is involved in ribosomal subunit association. However, the two most important effects of L41 were on peptidyltransferase activity and translocation. Peptidyltransferase activity was measured as a second-order rate constant (k(cat)/K-s) corresponding to the rate of peptide bond formation; this k(cat)/K-s was lowered 3-fold to 1.15 min(-1) mM(-1) in the L41 mutant compared with 3.46 min(-1) mM(-1) in the wild type. Translocation was also affected by L41. Elongation factor 2 (EF2)-dependent (enzymatic) translocation of Ac-Phe-tRNA from the A- to P-site was more efficient in the absence of L41, because 50% translocation was achieved at only 0.004 muM EF2 compared with 0.02 muM for the wild type. Furthermore, the EF2-dependent translocation was inhibited by 50% at 2.5 muM of the translocation inhibitor cycloheximide in the L41 mutant compared with 1.2 muM in the wild type. Finally, the rate of EF2-independent (spontaneous) translocation was increased in the absence of L41.