The available techniques do not permit the measurement of hepatocellular function, cardiac output, effective blood volume, and oxygen saturation in small animals without the need of blood or tissue sampling. We present methodology that permits us to measure the above variables in the rat. Multiple doses of indocyanine green (ICG; 0.16-2.6 mg/kg) were given to pentobarbital-anesthetized rats, and [ICG] was continuously measured by an in vivo hemoreflectometer. Initial velocity of clearance (V(0)) was determined from the [ICG] vs. time plot for each dose by nonlinear regression. The maximal velocity of clearance (V(max); indicating hepatocellular function) was calculated. V(max) was 1) accurately determined using two, three, or four doses of ICG; 2) it was 1.09 ± 0.16 mg·kg-1·min-1, not different from the V(0) of the near-saturating dose of 25 mg/kg; and 3) it decreased after partial hepatectomy. Cardiac output was 41.89 ± 1.46 ml·100 g-1·min-1, effective blood volume was 6.12 ± 0.34 ml/100 g, and oxygen saturation was 92 ± 2%. These methodologies may prove useful in the evaluation of hepatocellular dysfunction and hemodynamic parameters in the rat during and after various diseased states.