Recent studies on endogenous SsrA-tagged proteins have revealed that the tagging could occur at a position corresponding to the normal termination codon. During the study of SsrA-mediated LacI tagging (Abo et al., EMBO J, 2000 19:3762–3769), we found that a variant LacI (LacIΔC1) lacking the last C-terminal amino acid residue is efficiently tagged in a stop codon-dependent manner. SsrA tagging of LacIΔC1 occurred efficiently without LacI binding to the lac operators at any one of three stop codons. The C-terminal (R)LESG peptide of LacIΔC1 was shown to trigger the SsrA tagging of an unrelated protein (CRP) when fused to its C terminus. Mass spectrometry analysis of the purified fusion proteins revealed that SsrA tagging occurs at a position corresponding to the termination codon. The alteration of the amino acid sequence but not the nucleotide sequence of the C-terminal portion eliminated the tagging. We also showed that the tagging-provoking sequences cause an efficient translational readthrough at UGA but not UAA codons. In addition, we found that C-terminal dipeptides known to induce an efficient translation readthrough could cause an efficient tagging at stop codons. We conclude that the amino acid sequence of nascent polypeptide prior to stop codons is a major determinant for the SsrA tagging at all three stop codons.