A protein that binds to the sterol regulatory region of the hamster promoter for 3-hydroxy-3-methylglutaryl-coenzyme A reductase has been identified. All of the DNA bases crucial to the binding of this protein were previously shown to be essential for sterol regulation of the intact promoter in cultured cells. This low abundance protein, called Red 25, has been purified from nuclear extracts of hamster liver by a series of standard chromatographic techniques coupled with a DNA affinity step. Its size has been estimated as approximately 42 kDa by gel electrophoresis, size exclusion chromatography, and protein-DNA cross-linking studies. Furthermore, it binds to its target site with a Kd = 6 x 10(-11) M. Red 25 does not bind to the sterol regulatory regions of the LDL receptor or 3-hydroxy-3-methylglutaryl-coenzyme A synthase. This is consistent with recent studies that show there is a unique site for sterol regulation in the reductase promoter. The identification and purification of this protein represents a significant step in the study of feedback regulation by cholesterol.