Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis. Two ACAT genes exist in mammals. We report here the genomic organization of human ACAT-2 gene and analysis of its promoter activity in various cell lines. The human ACAT-2 gene spans over 18 kb and contains 15 exons, Three transcription start sites and one poly(A) site are identified by the 5'/3'-RACE. In addition, the human ACAT-2 gene is linked to the insulinlike growth factor binding protein 6 (IGFBP-6) gene in a head-to tail manner with a small intergenic region of about 1.2 kb. The 5'-flanking region of human ACAT-2 gene contains many potential cis-acting elements for multiple transcriptional regulatory factors but lacks TATA and CCAAT boxes. Using promoter-luciferase reporter assays, we demonstrate the transcriptional activity of ACAT-2 gene promoter is high in Caco-2 cells, especially after these cells become postconfluent and behave as intestinal enterocytes.