The nondegenerate two-photon absorption spectrum of all-traps retinal in a nonpolar solvent at room temperature has been measured for the first time. The multichannel detection utilizing the technique of femtosecond time-resolved pump-probe absorption spectroscopy has enabled us to obtain precise two-photon absorption spectra. It was found that the two-photon absorption maximum of all-traps retinal is located at 376 nm, which is 7 nm longer than the one-photon absorption peak. This peak wavelength difference indicates the 500-cm(-1) energy gap between the S-3 (B-u) state and the S-2 (A(g)) state. On the basis of the obtained two-photon absorption data and the available fluorescence data, we determined the 0-0 position of the S-2 state to be 2.47 x 10(4) cm(-1). (C) 2003 Elsevier B.V. All rights reserved.