The expression of human squalene synthase (HSS) gene is transcriptionally regulated in HepG-2 cells, up to 10-fold, by variations in cellular cholesterol homeostasis. An earlier deletion analysis of the 5'-flanking region of the HSS gene demonstrated that most of the HSS promoter activity is detected within a 69-base pair sequence located between nucleotides -131 and -200. ADD1/SREBP-1c, a rat homologue of sterol regulatory element-binding protein (SREBP)-1c binds to sterol regulatory element (SRE)-1-like sequence (HSS-SRE-1) present in this region (Guan, G., Jiang, G., Koch, R. L. and Shechter, I. (1995) J. Biol. Chem. 270, 21958-21965). In our present study, we demonstrate that mutation of this HSS-SRE-1 element significantly reduced, but did not abolish, the response of HSS promoter to change in sterol concentration. Mutation scanning indicates that two additional DNA promoter sequences are involved in sterol-mediated regulation. The first sequence contains an inverted SRE-3 element (Inv-SRE-3) and the second contains an inverted Y-box (Inv-Y-box) sequence. A single mutation in any of these sequences reduced, but did not completely remove, the response to sterols. Combination mutation studies showed that the HSS promoter activity was abolished only when all three elements were mutated simultaneously. Co-expression of SRE-1- or SRE-2-binding proteins (SREBP-1 or SREBP-2) with HSS promoter-luciferase reporter resulted in a dramatic increase of HSS promoter activity. Gel mobility shift studies indicate differential binding of the SREBPs to regulatory sequences in the HSS promoter. These results indicate that the transcription of the HSS gene is regulated by multiple regulatory elements in the promoter.