A technique has been established to determine the first hyperpolarizability value beta of fluorescent molecules based on femtosecond hyper-Rayleigh scattering (HRS). The intrinsic high harmonic content of the femtosecond pulse train is used as a high frequency amplitude modulation source. Due to the nonzero fluorescence lifetime, a high amplitude modulation frequency will result in an amplitude demodulated and phase shifted fluorescence signal. At very high modulation frequencies, the fluorescence signal becomes completely demodulated and only the inherent HRS signal will remain. A comparative study was made between the dispersion free beta(0)-value of a nonfluorescent ionic hemicyanine dye (DASPC(22)(+)) and the dispersion free beta(0)-value of the fluorescent inclusion complex of the dye incorporated in an amylose matrix. The fluorescence-free beta(0)-value for the complex appeared to be twice [(200 +/- 5) x 10(-30) esu] the beta(0)-value of the dye itself [(100 +/- 10) x 10(-30) esu]. (C) 1999 Elsevier Science B.V. All rights reserved.