In order to investigate the role of the hormone-sensitive lipase (HSL) gene in the activity of neutral cholesterol ester hydrolase (NCEH) from a molecular perspective, Chinese hamster ovary (CHO) cells were transfected with rat antisense hormone-sensitive lipase cDNA, and three different cell lines, designated as anti-HSL, were established. NCEH activity in anti-HSL cells was reduced to approximately 50% of that in control CHO cells. The concentration of cellular esterified cholesterol increased and the concentration of free cholesterol decreased in the anti-HSL cell lines. These results suggest that the HSL gene has a function of NCEH as well.