Peptide bond formation by the ribosome requires 23S rRNA and its interaction with the 3'-CCA end of tRNA. To investigate the possible evolutionary development of the peptidyl transfer reaction, we tried to obtain peptide bond formation without the ribosome or rRNA simply by using a piece of tRNA--an aminoacyl-minihelix--mixed with sequence-specific oligonucleotides that contained puromycin. Peptide bond formation was detected by gel electrophoresis, TLC analysis, and mass spectrometry. Peptide synthesis depended on sequence complementarity between the 3'-CCA sequence of the minihelix and the puromycin-bearing oligonucleotide. However, proximity of the reacting species was not by itself sufficient for peptide bond formation. In addition, imidazole as a catalyst was required. Its role may be similar to the recently proposed mechanism, wherein A2451 of 23S rRNA works as a general base. Thus, peptide bond formation can be achieved with a simple, minimized system that captures the essence of an interaction seen in the ribosome.