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Changes in the fluorescein fluorescence emission and excitation polarization spectra in synchronized cultured S3 fibroblasts at G1, mid-S, and mitosis, as well as in human lymphocytes before and after stimulation with mitogens, were studied. In contrast to those measured in aqueous solutions the emission and excitation polarization spectra in living cells exhibit a wavelength dependence characteristic for the state of the cell cycle. Changes in the temperature and in the amount of intracellular water result in quantitative wavelength-independent changes in the polarization spectra. Possible mechanisms for the qualitative wavelength-dependent changes in the fluorescein emission and excitation polarization spectra during the cell cycle are discussed.