Scavenger receptor BI influences multiple aspects of cellular sterol metabolism. In this series of studies, we evaluated the effect of scavenger receptor BI expression on the distribution and movement of sterol between the plasma membrane and the endoplasmic reticulum in macrophages, by comparing control J774 cells to J774 cells in which SR-BI expression was constitutively increased 3-fold. J774 cells with increased expression of SR-BI (J774-SRBI cells) esterified plasma membrane cholesterol more rapidly as compared to control cells. The esterification of endogenously synthesized cholesterol was also more rapid in cells with increased SR-BI expression; this could be partially suppressed by removing cholesterol from the plasma membrane. The increased plasma membrane sterol esterification in J774-SRBI cells was not due to increased acyl-coA:cholesterol acyltransferase activity and was observed even though J774-SRBI cells manifested a smaller free cholesterol pool in the endoplasmic reticulum. Cholesterol ester hydrolysis was also more rapid in J774-SRBI cells. Increased expression of SR-BI also facilitated the clearance of cellular cholesterol ester to HDL₃. This latter observation, combined with the measurement of the smaller ER free cholesterol pool in J774-SRBI cells, suggests that the free cholesterol derived from the hydrolysis of cholesterol ester was rapidly transported back to the plasma membrane. It is concluded that expression of SR-BI in macrophages increases the rate of free cholesterol transport, and modulates free cholesterol distribution between the plasma membrane and the internal membrane compartments in macrophages.