This article describes results obtained when human liver cells obtained from reduced grafts are cultured in a chemically defined medium. Remnants of livers after reduction for pediatric transplantation were processed by a multiple cannulation system through the existing vasculature, which allowed the homogeneous perfusion of collagenase. The graft weight ranged between 55 and 1000 g (median value: 145.6 g). The yield ranged between 0.13 × 106 and 38 × 106 cells/g of tissue (median value 14.73 × 106 cells/g), and the viability was 61.17 ± 27.43%. The total number of cells ranged between 57.6 × 106 and 12 150 × 106 cells (median value: 740 × 106 cells). Cells were cultured for 30 days. Albumin synthesis was observed during the first 2 weeks, with a peak value at day 6 (27.85 ± 1.77 μg/mL). Urea production was detected during the first week (peak value at day 6: 17.12 ± 2.11 mg/dL). Light microscopy showed the presence of cells in a monolayer. Biliary pigments were observed at day 20. By immunohistochemistry, positive cells for albumin, for hepatocyte marker, cytokeratin 19, CD 34, CD 68, and for alpha actin for smooth muscle, were observed. Our results showed that hepatocytes obtained from reduced liver grafts are easily cultured and are able to maintain viability and functionality in vitro. This alternative source of human cells maintained under controlled culture conditions may play an important role in the development of a bioartificial liver.