Urea cycle (UC) is the main pathway of ammonium removal. A deficiency in any of the five classical enzymes of the pathway causes a urea cycle disorder. Hepatocellular transplantation is one of the techniques applicable to treat this disorder. In the present work, we investigated the activities and the relative expression levels of two of the UC enzymes: Carbamyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC), in isolated hepatocytes preserved up to 120 h in University of Wisconsin (UW) solution at 0 degrees C, and during the rewarming of these suspensions. During preservation, CPSI showed differences in mRNA levels respect to time 0, while ornithine transcarbamylase remained unchanged. At the end of the rewarming, CPSI showed values of enzymatic activity and relative mRNA level comparable with the control, meanwhile, there was an increment in OTC activity. In line with these results, we found that hepatocytes cold preserved up to 120h in UW solution maintained their ability to remove an ammonium load comparable to freshly isolated hepatocytes. These data indicated that our preservation conditions up to 120h in UW solution followed by rewarming, preserves UC enzymes at levels similar to freshly isolated hepatocytes, allowing the use of these cells in bioartificial liver devices or hepatocellular transplantation.