Rose bengal is a dye that is not biotransformed before excretion into the bile and can be used as a measure of hepatic excretory function. Rose bengal disappeared from the blood with time in a biexponential manner. Blood disappearance curves of rose bengal after doses between 0.01 and 10 mg/kg in the rat were almost identical, having an initial biological half-life of 2 min and a terminal half-life of 100 min. With higher doses, rose bengal disappeared from the blood at a much slower rate, apparently due to saturation of hepatic excretory processes. Rose bengal was excreted into bile by a mechanism that is consistent with a first-order process which exhibits a biological half-life of about 30 min unless a high dose that saturated the process was given. Since the terminal half-life of the plasma disappearance is much different than the biliary excretory half-life, the two compartment model cannot be used to quantitate hepatic uptake and biliary excretion of rose bengal. Species variation in the biliary excretion of rose bengal was observed. The biological half-life for excretion of rose bengal into the bile of rats and rabbits was similar (30 min), that of the guinea pig shorter (17 min), and that of the dog longer (46 min). Rose bengal clearance was slower in the bile duct-ligated rat, the rat with two-thirds of its liver removed, and in newborn rats, conditions which are known to alter hepatic excretory function. The blood concentration of rose bengal was higher in the two-thirds hepatectomized rats only during the first 30 min after administration and not at later times; thus, if rose bengal is used as a measure of hepatic function and if only one blood sample is taken, the time interval chosen is critical.