We demonstrate that the mRNA levels of glycerol-3-phosphate acyltransferase (GPAT), a mitochondrial enzyme catalyzing the initial step in glycerolipid synthesis, are induced during the differentiation of 3T3-L1 preadipocytes to adipocytes and following ectopic expression of rat adipocyte determination and differentiation factor 1 (ADD1), a protein with high homology to the human sterol regulatory element-binding protein-1 (SREBP-1). The increase in GPAT mRNA levels that occurs during differentiation is partially prevented by ectopic expression of a dominant negative form of ADD1. Nucleotide sequences corresponding to the proximal promoter of the murine mitochondrial GPAT gene (Jerkins, A. A., Liu, W. R., Lee, S., and Sul, H. S. (1995) J. Biol. Chem. 270, 1416-1421) bound SREBP-1a and NF-Y in electromobility shift assays. In addition, GPAT promoter-luciferase reporter genes were stimulated by co-expression of SREBP-1a. This increase was attenuated when either a dominant negative form of NF-Y was co-transfected into the cells or when the GPAT promoter contained mutations in the putative binding sites for SREBP-1a or NF-Y. These studies demonstrate that the regulated expression of the mitochondrial GPAT gene requires both NF-Y and ADD1/SREBPs. Thus, SREBPs/ADD1 regulate not only genes involved in cholesterol homeostasis and fatty acid synthesis but also a key enzyme in glycerolipid synthesis.