Extensive studies of parameters conditioning high plating efficiency of epithelial liver cells at primary seeding allowed us to set up a technique for the routine culture of liver cells from rats of various ages (18 day-old pc to 7 month-old) in Ham F10 medium supplemented with 10 p. cent fetal calf serum and 10 p. cent human serum. Cultures, after several passages, or sometimes at primary seeding were free of fibroblasts. Cell lines were kept for over one year with a subculture splitting ratio of 1 to 10 each week. The generation time of the cells was 16–18 hours. Caryotype analysis showed a high majority of normal diploid and tetraploid cells. Various enzymes and metabolic pathways have been studied in primary culture and in cell lines: enzymes of the anaerobic metabolism of hexoses and metabolism of steroid hormones. Activity glucose-6-phosphatase was nearly lost in all cultures. Aldolase showed a specific liver activity with a cleavage ratio of phosphofructoses (F-1,6-diP/-1-P) equal to 1 or about 1 in several primary cultures and cell lines. Many metabolites arising from incubation of cell lines with 14C-labelled corticosterone, corticosterone-21-sulfate, testosterone and progesterone have been isolated and quantitated by gas liquid chromatography (GC) and mass fragmentography coupled to GC, using 14C/12C isotope ratio measurements. These metabolites indicate the presence in cultured cells of 3α/β-steroid-reductases, 4-enesteroid-reductases, 17β-steroid-oxido-reductase, 11β-steroid-oxido-reductase and ring hydroxylases.