Very-low-density lipoproteins (VLDL) isolated from fresh normolipidemic plasma were able to compete with labeled low-density lipoproteins (LDL) for receptor binding as efficiently as LDL in human skin fibroblasts. However, VLDL from fresh plasma failed to stimulate acyl-CoA: cholesterol acyltransferase (ACAT) (EC 2.3.1.26) activity significantly. When plasma was stored at 4°C for 24–48 h before ultracentrifugation, the VLDL were able to stimulate ACAT activity significantly. Proteolytic and lipolytic enzymes do not appear to play a role in this phenomenon. The increased capacity of VLDL from stored plasma to stimulate ACAT activity appears to be due partly to the increase in the internalization and degradation processes. Both the larger and the smaller VLDL subclasses were able to stimulate ACAT activity in the cells. The actual changes that take place in the VLDL molecule during the storage of plasma are not known.