Enhanced cyan and yellow fluorescent proteins are widely used for dual color imaging and protein-protein interaction studies based on fluorescence resonance energy transfer. Use of these fluorescent proteins can be limited by their thermosensitivity, dim fluorescence, and tendency for aggregation. Here we report the results of a site-directed mutagenesis approach to improve these fluorescent proteins. We created monomeric optimized variants of ECFP and EYFP, which fold faster and more efficiently at 37 degrees C and have superior solubility and brightness. Bacteria expressing SCFP3A were 9-fold brighter than those expressing ECFP and 1.2-fold brighter than bacteria expressing Cerulean. SCFP3A has an increased quantum yield (0.56) and fluorescence lifetime. Bacteria expressing SYFP2 were 12 times brighter than those expressing EYFP(Q69K) and almost 2-fold brighter than bacteria expressing Venus. In HeLa cells, the improvements were less pronounced; nonetheless, cells expressing SCFP3A and SYFP2 were both 1.5-fold brighter than cells expressing ECFP and EYFP(Q69K), respectively. The enhancements of SCFP3A and SYFP2 are most probably due to an increased intrinsic brightness (1.7-fold and 1.3-fold for purified recombinant proteins, compared to ECFP & EYFP(Q69K), respectively) and due to enhanced protein folding and maturation. The latter enhancements most significantly contribute to the increased fluorescent yield in bacteria whereas they appear less significant for mammalian cell systems. SCFP3A and SYFP2 make a superior donor-acceptor pair for fluorescence resonance energy transfer, because of the high quantum yield and increased lifetime of SCFP3A and the high extinction coefficient of SYFP2. Furthermore, SCFP1, a CFP variant with a short fluorescence lifetime but identical spectra compared to ECFP and SCFP3A, was characterized. Using the large lifetime difference between SCFP1 and SCFP3A enabled us to perform for the first time dual-lifetime imaging of spectrally identical fluorescent species in living cells.