The present studies describe an unexpected action of a cholesteryl ester hydrolase inhibitor on MA-10 Leydig tumor cells. These studies were initially intended to use the inhibitor, diethylumbelliferyl phosphate, to block cholesteryl ester hydrolysis and, thus, determine the contributions of this form of cholesterol to steroidogenesis and reveal any direct hormone effects on cholesterol esterification. Although this compound acted as an effective inhibitor of the cholesteryl ester hydrolase in intact MA-10 cells, it inhibited steroidogenesis at lower concentrations and to a greater extent than could be explained by simple inhibition of the ester hydrolase enzyme. This compound proved not to be generally toxic, but blocked some process occurring between cAMP formation and cholesterol side-chain cleavage. The diethylumbelliferyl phosphate block of steroidogenesis was readily bypassed by 22-hydroxycholesterol. These data indicated that the compound inhibited cholesterol transport. The lesion in cholesterol transport was not general, but very specific; cholesterol translocation to the mitochondrial site of cholesterol side-chain cleavage was blocked by this organophosphate compound.