To study the role of nuclear regulatory proteins in mediating dietary effects, hepatic CCAAT/enhancer binding protein (C/EBP), mRNA and transcription rate were measured for C/EBP-α and C/EBP-β in nutritional states that profoundly alter energy metabolism and growth. Weanling male Sprague-Dawley rats were fed riboflavin-sufficient (R+) or deficient (R-) diets for 4 wk. A diet-restricted, pair-fed (RP) group was maintained concurrently, because riboflavin-deficient rats voluntarily decrease food consumption by ~50% compared with controls. Half of each group was deprived of food for 48 h. The 4-wk treatment altered hepatic levels of both proteins (P < 0.05). C/EBP-α protein levels were increased -twofold by diet restriction. C/EBP-β protein levels were increased nearly threefold by riboflavin deficiency. Starvation had no significant effect on the expression of either protein. We investigated the mechanism responsible for increased protein by measuring steady-state mRNA levels and transcription rates for C/EBP-α and C/EBP-β. In both isoforms, increases in mRNA were parallel to increases in transcription rates. The nutrient-induced changes in protein, mRNA and transcription rates could not be attributed only to alterations in serum glucagon or insulin concentrations. We conclude that 1) C/EBP-α and C/EBP-β expression responds to diet but may involve different dietary signals for diet restriction vs. riboflavin deficiency; 2) the dietary regulation of C/EBP-α and C/EBP-β expression seems to be controlled in part at the level of gene transcription; and 3) C/EBP-α and C/EBP-β nuclear proteins, by virtue of their increased quantities, may participate in regulating altered energy metabolism and growth by influencing hepatic transcription of key metabolic enzymes.