The red fluorescent protein Rtms5(H146S) displays a transition from blue (absorbance lambda(max) 590 nm) to yellow (absorbance lambda(max) similar to 453 nm) upon titration to low pH. The pK(a) of the reaction depends on the concentration of halide, offering promise for new expressible halide sensors. The protonation state involved in the low pH form of the chromophore remains, however, ambiguous. We report calculated excitation energies of different protonation states of an RFP chromophore model. These suggest that the relevant titration site is the phenoxy moiety of the chromophore, and the relevant base and conjugate acid are anionic and neutral chromophore species, respectively. (c) 2006 Elsevier B.V. All rights reserved.