We previously reported that sterol regulation of the rat fatty-acid synthase was lost when the DNA sequence between -73 and -43 of the promoter was deleted from a luciferase reporter construct (Bennett, M. K., Lopez, J. M., Sanchez, H. B., and Osborne, T. F. (1995) J. Biol. Chem. 270, 25578-25583). We also showed that there was a binding site for sterol regulatory element binding protein-1 (SREBP-1) in this region that contains a palindromic E-box motif (5'-CANNTG-3'). This is the consensus recognition element for basic-helix-loop-helix leucine zipper containing proteins such as the SREBPs. However, the SREBPs are unique basic-helix-loop-helix leucine zipper proteins that not only bind to a subset of E-boxes but also to the direct repeat SRE-1 element of the low density lipoprotein receptor promoter as well as to variant sites present in the promoters for key enzymes of both cholesterol and fatty acid biosynthesis. Based on the sequence of the variant SREBP recognition sites in these other promoters, we noted there was more than one potential recognition site for SREBP within the -73 to -43 interval of the fatty-acid synthase promoter. In the present studies we have systematically mutated these potential SREBP sites and have analyzed the consequences on sterol regulation, activation by exogenously supplied SREBPs, and binding by SREBPs in vitro. The results clearly show that the E-box element is not the SREBP recognition site in this region. Rather, there are two independent SREBP binding sites that flank the E-box, and both are required for maximal sterol regulation and activation by transfected SREBP protein.