We analyzed molecular mechanisms controlling the activity of the nutritionally regulated promoter I (PI) of the bovine acetyl-CoA-carboxylase-alpha gene. PI-activity is driven by NF-Y and C/EBP factors having bound to high affinity sites on the proximal PI. However, both factors may also bind to low affinity sites on the distal PI. From there, they act as repressors of PI. We found that NF-Y may diminish and block C/EBP-beta mediated PI activation, even in trans, through direct factor interaction. Alterations in the concentrations of both factors apparently regulate the tissue specific PI-activity.