Cholesterol is a key lipid in the stratum corneum (SC), where it is critical for permeability barrier homeostasis. The epidermis is an active site of cholesterol synthesis, but inhibition of epidermal cholesterol synthesis with topically-applied statins only modestly affects epidermal permeability barrier function, suggesting a possible compensatory role for extra-epidermal cholesterol. Scavenger receptor class B type I (SR-BI) is a recently described cell surface receptor for high density lipoproteins (HDL), which mediates the selective uptake of cholesterol esters from circulating HDL. In the present study, we demonstrate, using both Northern and Western blots, that SR-BI is present in cultured human keratinocytes, and that calcium-induced differentiation markedly decreases SR-BI levels. Immunofluorescence staining localized SR-BI expression to the plasma membrane of undifferentiated keratinocytes, but revealed very little signal in differentiated cells. Additionally, the uptake of 3H-cholesterol-labeled HDL decreased in differentiated vs. undifferentiated kereatinocytes. Furthermore, the inhibition of cholesterol synthesis with simvastatin resulted in a 3 to 4-fold increase in both SR-BI mRNA and protein levels, while conversely, addition of 25-hydroxycholesterol suppressed SR-BI levels by approximately 50%. SR-BI mRNA is also expressed in murine epidermis, increasing by 50% in parallel with cholesterol requirements following acute barrier disruption. Since the increase is completely blocked by occlusion with a vapor-impermeable membrane, changes in epidermal SR-BI expression are regulated specifically by barrier requirements. Lastly, using immunofluorescence we demonstrated that SR-BI is present in human epidermis predominantly in the basal layer and increases following barrier disruption. In summary, the present study demonstrates first, that SR-BI is expressed in keratinocytes and regulated by cellular cholesterol requirements, suggesting that it plays a role in keratinocyte cholesterol homeostasis. Second, the increase in SR-BI following barrier disruption suggests that SR-BI expression increases in order to facilitate cholesterol uptake leading to barrier restoration.