Objective To find out the optimal procedures and suitable hepatocytes for the extracorporeal bioartificial liver support system (EBLSS), we observed the differences in bilirubin metabolism between fresh-separated hepatocytes and pre-incubated cells for 24 hours from adult animals Methods Hepatocytes were obtained by mechanical separation from male mice of 3 months age, suspended in high glucose DMEM culture medium containing 20% fetal bovine serum and 5 U/ml insulin, and divided into fresh-separated and pre-incubated groups. For hepatocytes in fresh-separated group, they were cultured immediately after separation in medium containing bilirubin serum for 3 hours. For hepatocytes of pre-incubated group, the cells were first cultured for 24 hours, and then cultured for 3 hours in medium containing bilirubin serum. Bilirubin serum in medium without hepatocytes was used as the control.Total, direct and indirect bilirubin concentrations were determined by using an auto-analyzer (Backman). Determinations were repeated in triplicate. Results Total bilirubin and direct bilirubin reduced by 33.91% and 39.29%, respectively, in fresh-separated group, and reduced by 8.0% and 5.40%, respectively, in pre-incubated group. There were statistically differences in changes of total bilirubin (P＜0.01, t =3.707) and indirect bilirubin(P＜0.01, t = 4.734) between the two groups. Conclusion Fresh-separated hepatocytes from adult animals have the better activity of bilirubin metabolism than the pre-incubated cells, and are recommended in EBLSS.