Macrophage foam cells must accommodate continuing fluxes of free cholesterol in spite of a greatly expanded store of cholesteryl ester. Though endogenous free cholesterol synthesis is suppressed, free cholesterol continues to enter the cell via endocytosis of oxidized/modified lipoproteins. It has been shown previously that this free cholesterol is released into the lysosomal compartment and rapidly transported to the plasma membrane prior to its esterification. A substantial amount of free cholesterol is also presented via the continuous hydrolysis of cholesteryl ester during the cholesteryl ester cycle. We addressed the question of whether the intracellular free cholesterol derived from the hydrolysis of cholesteryl ester formed a protected pool for rapid re-esterification. Incubation of macrophage foam cells with cyclic AMP to enhance cholesteryl ester hydrolysis, and with S58035 to inhibit acyl-CoA:cholesterol acyltransferase (ACAT) activity, led to conversion of cellular cholesteryl ester to free cholesterol and transport of this free cholesterol to the plasma membrane. Addition of progesterone, previously demonstrated to be an inhibitor of free cholesterol transport in other cell types, also led to conversion of cholesteryl ester to free cholesterol even though progesterone was only a weak inhibitor of ACAT activity. Free cholesterol in the plasma membrane was an important source of ACAT substrate to balance the constitutive hydrolysis of cholesteryl ester in cholesterol-loaded macrophages. Treatment of cells with progesterone, however, prevented free cholesterol derived from cholesteryl ester hydrolysis from moving to the plasma membrane. The sequestration of free cholesterol by progesterone could be reversed by incubation with human HDL3.(ABSTRACT TRUNCATED AT 250 WORDS)