The ribosome is a ribozyme. However, in bacterial ribosomes, the N-terminus of L27 is located within the peptidyl transfer center. The roles of this protein in real time remain unclear. We present single-molecule fluorescence resonance energy transfer (FRET) studies of tRNA dynamics at the peptidyl transfer center in ribosomes containing either wild type (WT) L27, or L27 mutants with A2H3, A2H3K4 or nine N-terminal residues removed. Removing L27's first three N-terminal residues or mutating a single residue, K4, reduces the formation of a stable peptidyl tRNA after translocation. These results imply that L27 stabilizes the peptidyl tRNA and residue K4 contributes significantly to the stabilization.