We studied the emission of mutants of the red fluorescent protein DsRed by room temperature single molecule fluorescence spectroscopy. Bulk samples of the DsRed variant E8 show mixed green and red fluorescence of equivalent intensities individually spectrally similar to arrested green and mature red fluorescent forms of DsRed. Investigations at the single molecule level indicate that, like DsRed, E8 is not monomeric at single molecule concentrations. The entities visualized are composed of green and red emitting proteins without a fixed ratio of green to red fluorescing units. We find indications for only weak, if any, fluorescence resonance energy transfer (FRET) between red and green chromophores within one E8 entity. (C) 2002 Elsevier Science B.V. All rights reserved.