The regulation of sterol regulatory element-binding proteins (SREBP) by cholesterol flux was studied in the intestinal cell line CaCo-2. CaCo-2 cells were incubated for 18 h with micelles containing 5 mM taurocholate and 500 microM oleic acid or micelles containing either 200 microM cholesterol or 150 microM lysophosphatidylcholine. In some incubations, an ACAT inhibitor was added or 25-hydroxycholesterol was substituted for cholesterol. The SREBP-1a transcript was 2-fold more abundant than the SREBP-1c transcript. In cells incubated with micelles containing cholesterol, rates of cholesterol synthesis were decreased and rates of esterification were increased. Cholesterol synthesis was decreased further by ACAT inhibition. Cholesterol influx decreased mRNA levels of SREBP-2, HMG-CoA synthase, HMG-CoA reductase, and fatty acid synthase. ACAT inhibition modestly suppressed gene expression further. Neither SREBP-1a nor SREBP-1c mRNA levels were altered by cholesterol. Despite decreases in gene expression of the sterol-responsive genes by cholesterol, the amounts of precursor and mature forms of SREBP-1 and SREBP-2 were not altered. In contrast, if 25-hydroxycholesterol was substituted for cholesterol, both the precursor and mature forms of SREBP-2 were decreased. The polar sterol decreased the mature form of SREBP-1 but the amount of the precursor form was unchanged. In cells incubated with micelles containing lysophosphatidylcholine, which causes cholesterol to efflux from cells, sterol-responsive gene expression was increased. The amounts of precursor and mature forms of SREBP-1 and SREBP-2, however, were not altered. In contrast, if the cells were depleted of cholesterol by incubating them with lovastatin and cyclodextrin, the mature forms of SREBP-1 and SREBP-2 were increased, as were mRNA levels for the sterol-responsive genes. The data would suggest that cholesterol influx/efflux regulates mRNA levels of sterol-responsive genes independently of changes in the amount of mature SREBP. In contrast, 25-hydroxycholesterol influx or cholesterol depletion alters the amount of mature SREBP, leading to the regulation of sterol-responsive gene expression.