A monomeric red fluorescent protein (mRFP1) was developed from the tetrameric red fluorescent protein DsRed through directed evolution. DsRed, originally a tetramer, was modified by introducing arginine residues at key interfaces to disrupt its quaternary structure. This process led to the creation of a dimer, and further mutations resulted in a true monomer, mRFP1. mRFP1 matures more rapidly than DsRed, showing similar brightness in living cells. It has excitation and emission peaks at 584 and 607 nm, which are 25 nm red-shifted from DsRed, enhancing tissue penetration and spectral separation from autofluorescence. Although mRFP1 has lower extinction coefficient, quantum yield, and photostability than DsRed, it is more suitable for use in genetically encoded fusion tags due to its monomeric nature. mRFP1 was tested in mammalian cells and showed functional gap junctions when fused with the gap junction protein Cx43, unlike tetrameric and dimeric forms. The development of mRFP1 represents a significant advancement in red fluorescent proteins, providing a valuable tool for cell biology and biotechnology. The study highlights the importance of directed evolution in overcoming the challenges of oligomeric fluorescent proteins and demonstrates the potential of mRFP1 as a superior alternative to DsRed in various applications.A monomeric red fluorescent protein (mRFP1) was developed from the tetrameric red fluorescent protein DsRed through directed evolution. DsRed, originally a tetramer, was modified by introducing arginine residues at key interfaces to disrupt its quaternary structure. This process led to the creation of a dimer, and further mutations resulted in a true monomer, mRFP1. mRFP1 matures more rapidly than DsRed, showing similar brightness in living cells. It has excitation and emission peaks at 584 and 607 nm, which are 25 nm red-shifted from DsRed, enhancing tissue penetration and spectral separation from autofluorescence. Although mRFP1 has lower extinction coefficient, quantum yield, and photostability than DsRed, it is more suitable for use in genetically encoded fusion tags due to its monomeric nature. mRFP1 was tested in mammalian cells and showed functional gap junctions when fused with the gap junction protein Cx43, unlike tetrameric and dimeric forms. The development of mRFP1 represents a significant advancement in red fluorescent proteins, providing a valuable tool for cell biology and biotechnology. The study highlights the importance of directed evolution in overcoming the challenges of oligomeric fluorescent proteins and demonstrates the potential of mRFP1 as a superior alternative to DsRed in various applications.