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Liver cholestasis is known to accompany several major liver disorders and is adequately mimicked in rats by ligation of the bile duct (BDL). L-arginine is a semi-essential amino acid which is involved in several important metabolic pathways that are significantly affected during cholestasis. This study was conducted in order to contribute to the understanding of the enrolment of L-arginine supplementation in cholestatic liver function. This was carried out by estimation of serum and liver tissue arginase activity, along with liver tissue citrulline, nitric oxide (NO) and polyamine concentrations. Rats subjected to BDL were treated for nine days with L-arginine (150 mg/kg) or remained without any treatment. Animals from two control groups were either subjected to medial laparotomy (sham/opened group) or were without any surgical treatment and received only L-arginine. Application of L-arginine prevented a significant increase in plasma bile acid and bilirubin concentrations, as well as enzyme biochemical markers that were increased after BDL. It is worth mentioning that L-arginine was able to cause a decrease in arginase activity and liver tissue NO concentrations that were found to be significantly altered during cholestasis. On the other hand, the changes occurring in the concentration of liver polyamines (putrescine, spermidine and spermine) and the activity of polyamine metabolizing enzymes were not notably affected by the administered L-arginine. The results of the present study revealed that exogenous L-arginine was able to ameliorate or prevent changes occurring in its metabolism in liver during cholestasis.