AbstractLysine methylation is one of the important post‐translational modifications of histones, and produces an Nε‐mono‐, di‐, or trimethyllysine residues. Multiple and site‐specific lysine methylations of histones are essential to define epigenetic statuses and control heterochromatin formation, DNA repair, and transcription regulation. A method was previously developed to build an analogue of Nε‐monomethyllysine, with cysteine substituting for lysine. Here, we have developed a new method of preparing histones bearing multiple Nε‐monomethyllysine residues at specified positions. Release factor 1‐knockout (RFzero) Escherichia coli cells or a cell‐free system based on the RFzero cell lysate was used for protein synthesis, as in RFzero cells UAG is redefined as a sense codon for non‐canonical amino acids. During protein synthesis, a tert‐butyloxycarbonyl‐protected Nε‐monomethyllysine analogue is ligated to Methanosarcina mazei pyrrolysine tRNA (tRNAPyl) by M. mazei pyrrolysyl‐tRNA synthetase mutants, and is translationally incorporated into one or more positions specified by the UAG codon. Protecting groups on the protein are then removed with trifluoroacetic acid to generate Nε‐monomethyllysine residues. We installed Nε‐monomethyllysine residues at positions 4, 9, 27, 36, and/or 79 of human histone H3. Each of the Nε‐monomethyllysine residues within the produced histone H3 was recognized by its specific antibody. Furthermore, the antibody recognized the authentic Nε‐monomethyllysine residue at position 27 better than the Nε‐monomethyllysine analogue built with cysteine. Mass spectrometry analyses also confirmed the lysine modifications on the produced histone H3. Thus, our method enables the installation of authentic Nε‐monomethyllysines at multiple positions within a protein for large‐scale production.