The aim of this study was to investigate plasma membrane fluidity in human keratinocytes using fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its cationic derivative 1-[4-(trimethylamino)-phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Keratinocytes from normal or psoriatic skin were isolated using trypsin-EDTA or dispase. In keratinocytes isolated from normal skin, TMA-DPH anisotropy values were higher than those observed using DPH; the difference must be related to the different localization of the two probes. In fact, DPH in whole cells localizes in plasma as well as intracellular membranes, yielding an average value of fluidity, while the cationic derivative TMA-DPH resides in the plasma membrane of the whole cells for a sufficient time for anisotropy measurements. Moreover, it has to be considered that plasma membrane is more ordered than intracellular membranes. The kinetics of incorporation of TMA-DPH was similar in keratinocytes isolated using trypsin-EDTA and those isolated using; dispase, however, the fluorescence anisotropy values were lower in keratinocytes isolated with dispase (0.260 +/- 0.01 vs 0.270 +/- 0.01, p = 0.029). This difference is probably related to modifications of lipid-protein interactions after trypsin treatment. Since no damage to plasma membrane after incubation with dispase seems to have been reported, we decided to use this separation procedure to study plasma membrane fluidity in psoriasis, a human pathological condition characterized by excessive cell proliferation and incomplete differentiation. Lower anisotropy values (0.260 +/- 0.01 vs 0.270 +/- 0.01, p = 0.001), indicating an increase in fluidity, were observed in keratinocytes isolated from skin of psoriatic patients than in epidermal cells isolated from normal human skin. We suggest that the measurement of fluorescence anisotropy in living cells is a convenient and useful tool to study membrane fluidity in human keratinocytes isolated from normal and diseased skin. Its application represents a technical advance because plasma membrane fluidity can be measured using very limited amounts of tissue, as obtained from biopsies.