Our results highlight a hitherto unknown role for cortisol in acute stress adaptation that is nonspecific and involves changes in membrane fluidity. We demonstrate a rapid change in liver plasma membrane fluidity in response to stressed levels of cortisol in vitro. The fluidity changes seen with cortisol were not dose-related, but occurred above a certain threshold suggesting a receptor-independent mechanism likely associated with steroid incorporation into the lipid domain. This was supported by the inability of membrane impermeable ABT-199 Bcl-2 inhibitor cortisol-PEP to alter membrane fluidity. While changes in plasma membrane cholesterol levels alter lipid order that appears unlikely in the present case as membrane cholesterol remained unchanged in response to cortisol treatment. The cortisol-induced fluidization of liver plasma membrane appears to be steroid specific, as neither 17b-estradiol nor testosterone treatment showed a similar response in trout plasma membrane. This agrees with the recent findings that the chemical structure of the steroid backbone affect interaction with the lipid bilayer and subsequent changes in plasma membrane fluidity. It remains to be determined whether the membrane biophysical effect is also seen with other corticosteroids and not just cortisol. However, cortisol is the primary corticosteroid that is released into the circulation in response to stress in trout. The membrane fluidizing effect of cortisol seen in liver may be a generalized response affecting all tissues in response to stress. Mammalian studies reported a fluidizing effect of glucocorticoid on fetal rat liver and dog synaptosomal membranes, whereas an ordering effect was observed in rat renal brush border and rabbit cardiac muscle. This suggests that stress-mediated cortisol effect on membrane order may be tissue-specific, but this remains to be determined in fish. Altogether, our results indicate that stress-induced elevation in cortisol levels rapidly fluidizes liver plasma membrane in rainbow trout. AFM topographical and phase images further indicate that cortisol alters biophysical properties of liver plasma membranes. Specifically, cortisol exposure led to the reorganization of discrete microdomains, likely gel phase and disordered fluid-phase in the lipid bilayer. These discrete domains differed in height, which increased after cortisol treatment. A recent study on erythrocytes also reported a glucocorticoid-induced domain reorganization, which involved formation of large protein-lipid domains by hydrophobic and electrostatic interactions leading to alteration in membrane structure and elasticity. Similar domain changes have also been reported for synthetic lipids in response to halothane exposures or melting transitions, treatments that are known to increase membrane fluidity. Cortisol appears to have a greater effect on lower domains, as indicated by the greater change in surface adhesion following steroid treatment, compared to the higher lipid domains. Collectively, stressed levels of cortisol rapidly alter the biophysical properties of trout hepatic plasma membrane. We hypothesize that changes in membrane order by cortisol is the result of a non-uniform fluidization at the nanoscale among different membrane domains. Rapid changes to membrane order by cortisol may play a role in triggering acute stress-related signaling pathways. Indeed membrane order perturbations lead to rapid activation of cell signaling pathways, including protein kinases.