The turnover rate of an individual protein is a function of the rates of synthesis and loss of that protein. For most intracellular proteins, loss occurs through digestion by lysosomal or cytosolic proteases. Although a significant proportion of hepatic lysosomal enzymes is released from the hepatocyte by excretion into bile, the contribution of biliary excretion to the turnover of hepatic lysosomal enzymes has never been measured. Thus, we used in vivo pulse-labeling to determine the half-lives of two hepatic hydrolases, β-galactosidase (β-gal) and β-glucuronidase (β-glu). Each enzyme was purified by immunoisolation from hepatic lysosomes that were isolated at various times after injection of rats with 3H-labeled leucine. The decay curves for the specific radioactivities of β-gal and β-glu were used to calculate the half-lives of the proteins, which were 3.8 and 5.1 days, respectively. To determine the percent of total hepatic contents of each enzyme that was lost per day by biliary excretion, we collected bile from bile fistula rats for 24 hours and then used radioimmunoassays to quantitate the amounts of β-gal and β-glu in bile and liver samples of the same rats. We found that ∼4% of the total hepatic contents of both β-gal and β-glu was excreted into bile per day. Finally, we used these data to calculate that 31% and 41% of hepatic losses of β-gal and β-glu, respectively, were due to biliary excretion. These results suggest that extracellular release through biliary excretion is a major mechanism contributing to the turnover of lysosomal hydrolases.