Previous experiments have shown that the top of helix 90 of 23S rRNA is highly important for the ribosomal peptidyltransferase activity and might be part of the donor (P) site. Developing on these studies, mutations in the 23S rRNA at the highly conserved positions G2505, G2582, and G2583 were investigated. None of the mutations affected assembly, subunit association, or the capacity of tRNA binding to A and P sites. A “selective transpeptidation assay” revealed that the mutations specifically impaired peptide bond formation. Results with a modified “fragment” assay using the minimal donor substrate pA-fMet are consistent with a model where the nucleotides ΨGG2582 form a binding pocket for C75 of the tRNA.