The compatibility of beta-amino acids with ribosomal translation was studied for decades, but it has been still unclear whether the ribosome can accept various beta-amino acids, and whether the ribosome can introduce multiple beta-amino acids in a peptide. In the present study, by using the Escherichia coli reconstituted cell-free translation system with a reprogramed genetic code, we screened beta-amino acids that give high single incorporation efficiency and used them to synthesize peptides containing multiple beta-amino acids. The experiments of single beta-amino acid incorporation into a peptide revealed that 13 beta-amino acids are compatible with ribosomal translation. Six of the tested beta-amino acids (beta hGly, L-beta hAla, L-beta hPhg, L-beta hMet, and D-beta hPhg) showed high incorporation efficiencies, and seven (L-beta hLeu, L-beta hIle, L-beta hAsn, L-beta hPhe, L-beta hLys, D-beta hAla, and D-beta hLeu) showed moderate incorporation efficiencies; whereas no full-length peptide was produced using other beta-amino acids (L-beta hPro, L-beta hTrp, and L-beta hGlu). Subsequent double-incorporation experiments using beta-amino acids with high single incorporation efficiency revealed that elongation of peptides with successive beta-amino acids is prohibited. Efficiency of the double-incorporation of the beta-amino acids was restored by the insertion of Tyr or Ile between the two beta-amino acids. On the basis of these experiments, we also designed mRNA sequences of peptides, and demonstrated the ribosomal synthesis of peptides containing different types of beta-amino acids at multiple positions.