The role of the plasma membrane fluidity (PMF) on the shear sensitivity of HB-32 hybridomas to laminar fluid shear was investigated. Steady-state fluorescence anisotropy (rs) of the cationic fluorescent probe 1-[4-(trimethylamino) phenyl]-6-phenylhexa-1,3,5-triene, was used to evaluate the PMF of whole hybridoma cells. The PMF was manipulated by the addition of the anesthetic benzyl alcohol, by temperature changes and by cholesterol enrichment. The effect of these PMF modifying procedures on the shear sensitivity of HB-32 was assessed by exposing the cells to defined levels of laminar shear stress in a Couette flow device. Conditions that resulted in lower rs values (indicating higher PMF) yielded a more fragile cell. Batch cultivations supplemented with the shear protective agent Pluronic® F-68 showed higher values of rs compared to control experiments during various growth phases, suggesting that the protective mechanism of Pluronic F-68 relies on its ability to decrease the PMF through direct interaction with the plasma membrane. The protective mechanism of serum against turbulent fluid shear is also discussed from analysis of growth and death kinetics of agitated and static cultures at increasing serum levels. The results of this study show that the fluid state of the plasma membrane is important in determining the integrity of hybridomas when exposed to lethal shear levels. It is concluded that increasing membrane fluidity correlates with increasing shear sensitivity.