1. A new method to isolate and perfuse the rat spleen and liver simultaneously with a common blood perfusate at high haematocrit was developed. The spleen was pre-labelled with [3H]cholesterol, enabling reverse cholesterol transport from an extrahepatic tissue to the blood and thence to the liver and bile to be studied in a single preparation in vitro. 2. The presence of the liver significantly increased the release of [3H]cholesterol from the spleen by 15%, compared with experiments where the spleen was perfused alone. 3. There was a substantial release of [3H]cholesterol and cholesterol mass from the spleen to serum lipoproteins, the majority (80%) to high-density lipoprotein (HDL), in which cholesteryl ester accumulated. 4. The HDL subfractions HDL2 and HDL3 (d 1.085-1.250) were most important for removal of cholesterol from the spleen, whereas HDL1 and HDL2 (d 1.050-1.125) were important for delivery of cholesterol to the liver, a net uptake of cholesteryl ester occurring only from these fractions. 5. Approximately half of the [3H]cholesterol released by the spleen was recovered in erythrocytes. Also, in experiments utilizing a lipoprotein-free perfusate containing erythrocytes, a substantial quantity of [3H]cholesterol was transported and/or exchanged into the liver and bile, indicating that erythrocytes play an important role in the equilibration of unesterified cholesterol between the tissues.