In vitro studies indicate that bilirubin and other albumin-bound substances can be efficiently removed from plasma by filtration over albumin-conjugated agarose beads. The effectiveness of this technique in vivo was investigated in rats by using a closed extracorporeal hemoperfusion system. Five Gunn rats whose endogenous bilirubin pool had been labeled with [(3)H]bilirubin and five Sprague Dawley rats with surgically created biliary obstruction were chosen as models of unconjugated and conjugated hyperbilirubinemia. Indocyanine green was injected into rats and its removal also studied. In the Gunn rats, 98% of the bilirubin was removed from plasma during the initial pass over the column as determined isotopically and chemically. Plasma bilirubin levels fell more than 70% from 8.2+/-1.6 mg/100 ml (mean+/-SD) to 2.6+/-0.5 mg/100 ml during a 1-h hemoperfusion. An average of 1,061 mug of bilirubin was recovered from the columns, representing 22.5+/-4.2% of the total exchangeable bilirubin pool and 96+/-36.4% of the plasma pool. Results were similar in the rats with biliary obstruction and in those given indocyanine green. Normal Sprague Dawley rats experience minimal changes in formed blood elements, electrolytes, and proteins as the result of hemoperfusion. When the total volume of the column did not exceed 51% of the estimated blood volume of the animal, the survival rate was 100% in 20 studies, and the procedure was without observable ill effect. Extrapolation of both in vitro and in vivo data to man suggests that extracorporeal hemoperfusion over albumin-agarose columns may be a practical means of assisting hepatic excretory function.