Adrenals express a high level of neutral cholesteryl ester hydrolase (CEH) activity, and male rats have greater activity than females; however, the identity of the enzyme(s) responsible for this activity and the basis for the sex differences are unknown. Using mice in which hormone-sensitive lipase (HSL) was inactivated by homologous recombination (HSL −/−), neutral CEH activity was reduced more than 98% compared with controls. Female HSL −/− mice showed a reduction in stimulated corticosterone values. Mechanical separation of rat adrenals revealed less HSL in the outer than the inner cortex. Examination of subfractions of rat adrenals showed that immunoreactive HSL was prominently expressed in microsomes, with lesser amounts in the cytosol and little to no HSL in mitochondrial and nuclear fractions or the lipid droplet. Four- to 10-fold more neutral CEH activity was in the microsomal fraction than any other fraction. No sex differences in the expression or subcellular distribution of HSL protein were found; however, neutral CEH activity was lower in the microsomal fraction of females, and female adrenals contained more cholesteryl esters. Thus, HSL appears to be responsible for most, if not all, of adrenal neutral CEH activity, is prominently expressed in microsomes, and its activity is influenced by sex.