This study investigates the molecular mechanism of promoter I (PI) controlling the bovine acetyl-CoA-carboxylase-alpha (ACC-α) encoding gene. To this end, we proofed that PI is the prominent and regulated promoter of ACC-α in lipogenic tissues. PI is basically repressed by NF-Y and C/EBP factors binding to the 5´ distal region. However, joint over-expression of all three NF-Y factors or C/EBP factors enhances the promoter activity. The paradox was resolved by revealing that both factors bind also to high affinity sites in the proximal promoter area and strongly activate a minimal promoter construct. NF-Y factors diminish the potential of C/EBPβ to enhance the PI activity by protein-protein interaction. Endogenous assays revealed that the mRNA ratio of NF-Y over C/EBPβ correlates PI activity in a tissue-specific manner. A master control unit is proposed to modulate the PI activity in vivo.