The control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2; EC 2.7.1.105/3.1.3.46) gene expression during liver regeneration was studied. The level of PFK-2/FBPase-2 mRNA decreased to about 5% of the control value 6 hr after partial hepatectomy. Thereafter the mRNA increased to a maximum at 48 hr and returned to normal levels by 96 hr. In sham-operated animals, only a small increase was observed during the first 4 hr. The mRNA was recognized by a 299-base-pair liver-specific cDNA probe but not by a muscle-specific probe. The time course of mRNA modulation was well correlated with PFK-2/FBPase-2 activity and with the amount of bifunctional enzyme protein determined by immunoblotting with an antibody raised against the N-terminal decapeptide of liver PFK-2/FBPase-2. No alteration in the degradation rate of PFK-2/FBPase-2 mRNA was noted after partial hepatectomy. The modulation of PFK-2/FBPase-2 gene expression during liver regeneration involved changes in the transcription rate. The rate decreased by 50% at 6 hr after liver resection. The rate increased thereafter to a maximum at 72 hr and then returned to control values by 96 hr. The transcription rate of albumin did not change, whereas that of phosphoenolpyruvate carboxykinase increased 12-fold at 6 hr. These results show that PFK-2/FBPase-2 gene transcription is specifically regulated and that this regulation is in part responsible for the alterations in hepatic metabolism seen in regenerating liver.