The North American wood frog, Rana sylvatica, is one of the few vertebrate species that is capable of surviving freezing. Prevention of intracellular ice formation and maintenance of osmotic balance is facilitated by the production of large concentrations of urea during freezing in the liver. The regulatory roles of three enzymes critical to production of urea; glutamate dehydrogenase (GDH), carbamoyl phosphate synthetase I (CPS1), and ornithine transcarbamylase (OTC), were investigated in the liver in response to freezing. Freeze-exposed GDH had heightened substrate affinity and higher maximal activity than the control. Freeze-exposed GDH had decreased acetylation and ADP-ribosylation. CPS1 from frozen frogs was demonstrated to have a higher affinity for ammonia and decreased lysine glutarylation relative to the control. Freeze-exposed OTC demonstrated improved affinity for ornithine and increased serine phosphorylation relative. Taken together, the results suggest that urea production is sustained in the liver of the wood frog during freezing.