The kinetics of [4- 14C]cholesterol uptake by intima-media layers of thoracic aortas from normal rabbits have been studied by use of an in vitro method. 1. 1.The uptake of labelled cholesterol was followed during a 4-h period. The uptake curve shows an initial steep rise (estimated from the 15-min uptake value) followed by a step which has a less steep, obviously rectilinear course. 2. 2.The initial uptake of labelled cholesterol was found not to depend significantly on temperature (4–38°). The initial uptake is probably due to a superficial fixation of labelled cholesterol. 3. 3.The rate of [ 14C]cholesterol uptake after the initial 15 min is, on the contrary, temperature dependent, presumably due to two processes having different temperature dependences. One of the hypothetical processes is assumed to have a temperature optimum at 20°; the other has an “activation energy” of about 18 kcal/mole. Judging from the temperature dependence, the [ 14C]cholesterole uptake corresponding to the second phase of the uptake is thought to represent transport of cholesterol across the endothelial cell membrane. No causal explanation of the course of the uptake-temperature curve in the interval about 20° can be given at the present. 4. 4.The transfer rate of cholesterol from serum to intima-media is of the same order of magnitude as that found in vivo. 5. 5.The relationship between the rate of cholesterol transfer from serum to intima-media and total cholesterol concentration within the normal range (27 to 107 mg per 100 ml serum) is similar to the relationship between the velocity of an enzymatic reaction, showing substrate saturation phenomenon, and substrate concentration. In the model the initial uptake corresponds to the formation of enzyme substrate complex. Other interpretations of the relationship between cholesterol transfer rate and cholesterol concentration are discussed. 6. 6.Finally, the similarity of the kinetics found in this study with those for protein uptake by Ehrlich ascites-tumor cells and Amoeba proteus, assumed to be due to pinocytosis, is emphasized.