Growth of Chinese Hamster Ovary (CHO) cells in the presence of 20% lipid depleted serum (LDS) for only 2 hr results in an increase in the synthesis of [14C]sterols from [14C]mevalonate and from [14C]squalene compared with cells grown under normal growth conditions in the presence of 10% fetal calf serum (FCS). This enhanced sterol synthesis increases with time of exposure of the cells to LDS. However, exposing these cells for time periods up to 42.5 hr to a growth medium containing 20% LDS did not result in enhanced [14C]sterol synthesis from [14C]2,3-oxidosqualene. Incubation of these cells with [14C]mevalonate resulted in the accumulation of [14C]squalene regardless of the presence of either LDS or FCS. These results suggest that squalene epoxidase is a regulatory enzyme in the cholesterol biosynthetic pathway in CHO.