Mouse hepatocytes in primary culture were characterized. Hepatocytes were isolated by the two-step hepatic portal vein perfusion method described previously. An optimal cell attachment of 43% was noted after 2 h incubation in 10% fetal bovine serum. Minimal attachment (less than 7%) occurred in serumless medium. Serum concentrations above 10% and attachment durations greater that 2 h resulted in no increased attachment of viable cells. Nonviable cells, however, progressively attached when both of these parameters were increased. Survival data of the cells in culture resembled those reported for rat hepatocytes in primary culture. A progressive decrease in survival was noted following initial attachment until only approximately 15% of initially plated cells remained viable and attached after 8 d culture. The decrease in survival was accompanied by morphologic changes including flattening and elongation of the cells, some multinucleation, and disruption of monolayer groups.