The degree of depolarization of fluorescence light emitted from an organic dye, used as a molecular probe, is a powerful tool in probing the microenvironment. Polarization measurements of intracellular exogenous fluorescein have been shown to reflect the physiological state of the cells. The relationship between intracellular fluorescein fluorescence polarization (IFFP) and cell cycle, was investigated in the leukemia T-lymphocyte Jurkat cell line. Jurkat T cells were cultured in increasing cell densities, their cell cycle progression cytometrically monitored and the IFFP measured. At the highest cell density, the subpopulation of cells at the resting phases the (G₀/G₁) predominated, and the mean IFFP was 0.186 ± 0.015. At the lowest density, with diminished proportion of cells in the G₁/G₂ stages the mean IFFP decreased to 0.126 × 0.01. Treatment of the Jurkat T cell line with phase arrested agents 1 μM hydroxyurea, or 1 μM nocodazole, arrests the cells in the S and G₂/M phases, respectively. These treated cells exhibit significantly lower IFFP values, mean polarization value 0.140, as compared to 0.171 ± 0.009 in control cells. Preincubation of Jurkat cells in buffer results in accumulation of the cells in the G₀/G₁ phases as well as a parallel increase in IFFP. A characteristic decrease in IFFP was demonstrated upon triggering these cells with Phytohaemagglutinin (PHA). High correlation (Pearson correlation =0.942) was found between percentage of cells in the G0/G1 phases and the mean IFFP of the measured cell population. These results may indicate that the intracellular microviscosity of Jurkat T cells as measured by IFFP, is changing over the cell cycle.