VLCAD is a mitochondrial flavoenzyme encoded on the nuclear genome which catalyzes the first reaction in the fatty acid β-oxidation spiral. We sequenced 4 Kb of upstream 5'-flanking region of the human VLCAD gene. This promoter region lacks TATA or CAAT boxes and is extremely GC-rich. Chimeric plasmids containing various 5'-flanking genomic fragments fused upstream of the luciferase reporter gene were transiently transfected into human hepatoma cells, neonatal rat cardiomyocytes, mouse 3T3 fibroblast cells, and C2C12 myotube cells. The minimal promoter is 0.1 kb of the 5' flanking region. Transfection of constructs containing 4 kb, 2.3kb, 1.0kb, 0.3kb, 0.2kb, 0.16kb, and 0.12 kb of the 5'- flanking region activated VLCAD gene transcription activity form 10 to 30 fold as compared with transfection with the 0-.1 kb construct in HepG2 cells, and from 11 to 20 fold when compared with the 0.1 kb construct transfection in neonatal rat cardiomyocytes. Compared with a promoterless plasmid, these constructs activated luciferase activities from 276 to 482 fold in HepG2 cells and 45 to 95 in neonatal rat cardiomyocytes. These results indicate that the 20 bp between 0.12 kb and 0.1 kb contains important transcription factor binding sites. By computer search, there are two transcription factor consensus binding sites identified for AP-2 and IL-6 motifs in this region. We conclude that activation of VLCAD transcription is likely mediated by the 20 bp sequence, between 0.12 kb and 0.1 kb. We will search for the DNA binding factors activating thisβ-oxidation enzyme gene.