The accumulation of triglycerides in the liver has been associated with reduced hepatic function; however, direct evidence that fat accumulation causes decreased liver function is lacking. Hepatocyte monolayers isolated from ruminating calves with an initial low triglyceride concentration were either loaded or not loaded with triglycerides by incubation with 1.5 or 0 mM exogenous nonesterified fatty acids from 12 to 48 h after plating. Basal rates of synthesis of albumin and protein were not affected by triglycerides in the cell. Inclusion of insulin and glucagon from 12 to 72 h after plating increased rates of albumin and protein synthesis. Hepatocytes loaded with triglycerides were less sensitive to the hormonal stimulation of albumin and protein synthesis than were normal hepatocytes. Insulin clearance rates were also lower in hepatocytes loaded with triglycerides than in normal hepatocytes. Decreased insulin clearance and hormonal control of protein synthesis could contribute to the etiology of metabolic disorders that are associated with periparturient fatty liver in dairy cows.