Using 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) as a probe, we have done fluorescence anisotropy experiments to estimate, in situ, the temperature dependence of the lipid order of the plasma membrane of MDCK cells. In intact monolayers, anisotropy vs temperature biphasic curves were obtained, indicative of discontinuous change in lipid physical state starting around 27 degrees C, in the apical domain of the plasma membrane. The very high anisotropy values (r greater than 0.33) determined below this temperature strongly suggested it corresponded to a phase separation phenomenon. Biphasic curves with similar critical temperatures zone were also obtained when measuring anisotropy or fluorescence lifetime of suspensions of MDCK cells. This suggests that changes in lipid physical state might be involved in the marked dependence on temperature of tight junction formation and of electrical conductance in MDCK monolayers.