Radiolabeled 19-iodocholesterol is widely used to obtain images of human adrenals. We have shown in rats and mice that 85-95% of the radiolabel is present in the esterified form. Using rat adrenal cytosolic cholesterol esterase, the kinetic parameters K~t and V were determined for commercially available cholesteryl [l-14C]-oleate (!) and [125I]-19-iodocholesteryl oleate (It). The Kra and V were found to be 16.2/zM and 602 pmol/min/mg prot. respectively for I compared to 76.2 ttM and 37.6 pmol/min/mg prot. for II. Since V/Ku is 37.3 ml/min/mg prot. for the normal ester and 0.49ml/min/mg prot. for the iodinated analog, it appears that the normal substrate is 76 times more specific than II. In addition, direct competition experiments were run in which 67/~M I was used as substrate. The addition of 65 and 325/tM 19-iodocholesteryl oleate caused 20 and 49% inhibition, respectively. On the basis of these studies, it is not surprising that 19-iodocholesterol would accumulate in the adrenals in an esterified form and thus provide an effective agent for adrenal visualization.