The expression of the human SREBP-2 gene is transcriptionally regulated in a cooperative manner by sterol regulatory element-binding proteins (SREBPs) and the general transcription factor NF-Y [Sato, R., Inoue, J., Kawabe, Y., Kodama, T., Takano, T., and Maeda, M. (1996) J. Biol. Chem. 271, 26461-26464]. To understand the sterol-dependent transcriptional regulation by these factors in detail, we have examined the regulation of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and squalene synthase genes, whose promoters have multiple potential sterol regulatory elements (SRE, SREBP binding site) and NF-Y binding sites. The promoter of the human HMG CoA synthase gene was cloned, sequenced, and functionally characterized by means of reporter gene assays. The results indicate that an inverted CCAAT box, two SRE motifs and two Sp1 sites localized in a 90-bp region coordinately regulate the transcription. In the case of the human squalene synthase promoter, two SRE motifs and an inverted CCAAT box between the motifs localized in a 51-bp region are responsible for the sterol-regulated transcription of the gene. Gel mobility shift assay reveals that these two inverted CCAAT boxes are recognized by NF-Y. The involvement of multiple responsive elements in the transcription of HMG CoA synthase and squalene synthase seems to induce a higher level of sterol-dependent regulation (3.5 to 5. 8-fold) compared with that of the SREBP-2 promoter, which contains a single pair of SRE motif and CCAAT box (1.8 to 2.6-fold). Reporter gene assays using constructs containing various nucleotide spacing lengths between the SRE motif and the CCAAT box demonstrate that the 16 to 20-bp spacing range is required for maximal transcriptional regulation. These results agree with the findings that the distances between the two motifs in the known sterol responsive elements in several genes, including the human HMG CoA synthase and squalene synthase genes, are in this range.