High yields of human hepatocytes (up to 23 X 10(6) viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham's F12 containing 0.2% bovine serum albumin, 10(-8) M insulin, and 10(-8) M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250 +/- 177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50 +/- 0.17 nmol glucose.mg-1.min-1) similar to that reported for human liver. Insulin at 10(-8) M activated glycolysis (X1.40) and glycogenesis (X1.34), and glucagon at 10(-9) M stimulated gluconeogenesis (X1.35) and glycogenolysis (X2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein, haptoglobin, alpha 2-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10(-9) M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol.mg-1 cell protein.min-1, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol.mg-1). The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.