LCAD is a mitochondrial flavoenzyme encoded on nuclear genome which catalizes the first reaction in fatty acid beta-oxidation. We characterized 2 kb of upstream 5′-flanking region of the human LCAD gene by sequence analysis. This promoter region lacks TATA or CAAT boxes, is extremely GC-rich, contains SP1 binding sites, and has several nuclear response element (NRRE) consensus hexamers. Chimeric plasmids containing various 5′-flanking genomic fragments fused upstream of the chloramphenicol acetyl transferase(CAT) reporter gene were transfected into human hepatoma cells, neonatal rat cardiomyocytes, and mouse 3T3 fibroblast cells. Minimal promoter is -200 to +1 upstream of the Cap site. Two suppressor sequences, at -288 to -259, and -1048 to -1010 respectively, both contain two NRRE consensus binding sites, and interact with COUP-TF on gel shift analysis. To further define the promoter region of the human LCAD gene by transgenic mouse analysis, constructs containing the LCAD 5′-flanking regions, -1,800/+1 and -200/+1, linked with luciferase reporter gene excised from pT3/T7-LUC luciferase expression vector were micro-injected into mouse oocytes by standard techniques. Founder animals incorporating the LCAD-luciferase chimeric gene identified by PCR and tail DNA blot experiments were bred with normal C57BL/6J mice. F1 mice positive for the incorporated LCAD-luciferase fusion gene have been tested for expression by luciferase assay from various tissue extracts, and immunohistochemistry. Distribution of luciferase activity in various tissues of progeny of the human LCAD -200 or -1800/ luciferase indicated LCAD -200/+1 region contains an active promoter with some tissue specification; -1800/+1 region contains suppressor element. Thus, both in vitro andin vivo studies showed that LCAD NRRE-281 and LCAD NRRE-1032 contain suppressor activity, and LCAD -200/+1 contains promoter function with some tissue specificity. Funded by NIH grant #PO1-DK33487.