Sterol regulatory element-binding proteins (SREBPs) activate genes of cholesterol and fatty acid metabolism. In each case, a ubiquitous co-regulatory factor that binds to a neighboring recognition site is also required for efficient promoter activation. It is likely that gene- and pathway-specific regulation by the separate SREBP isoforms is dependent on subtle differences in how the individual proteins function with specific co-regulators to activate gene expression. In the studies reported here we extend these observations significantly by demonstrating that SREBPs are involved in both sterol regulation and carbohydrate activation of the FAS promoter. We also demonstrate that the previously implicated Sp1 site is largely dispensable for sterol regulation in established cultured cells, whereas a CCAAT-binding factor/nuclear factor Y is critically important. In contrast, carbohydrate activation of the FAS promoter in primary hepatocytes is dependent upon SREBP and both the Sp1 and CCAAT-binding factor/nuclear factor Y sites. Because 1c is the predominant SREBP isoform expressed in hepatocytes and 1a is more abundant in sterol depleted established cell lines, this suggests that the different SREBP isoforms utilize distinct co-regulatory factors to activate target gene expression.