Two-photon absorption spectra of 4'-hydroxybenzylidene-2,3-dimethylimidazolinone, a model chromophore of enhanced green fluorescent protein (eGFP), were measured in various solvents. The two-photon absorption band of its anionic form is markedly blue-shifted from the corresponding one-photon absorption band in all solvents. Moreover, the magnitude of the blue shift varies largely depending on the solvent, which does not accord with the assignment of the two-photon absorption band to the transitions to the vibrationally excited S-1 state. Our finding is readily rationalized by considering overlapping contributions of the S-1 <- S-0 and S-2 <- S-0 transitions, suggesting the involvement of the S-2 state also in two-photon fluorescence of eGFP. (C) 2015 Elsevier B.V. All rights reserved.