Usingin situhybridisation we show that, in the rat adrenal. 11β-hydroxylase is confined to the inner zones, whereas aldosterone synthase is expressed exclusively in the glomerulosa. Immunoblotting methods identify an 18-hydroxydeoxycorticosterone (18-OH-DOC) in IEF gels of solubilised inner adrenocortical zone membrane preparations. This steroid, which can also be identified by immunocytochemistry, cannot be solvent extracted from the IEF gels unless the gel slices are first treated with trypsin. Preincubation of viable whole glandular tissue with trypsin significantly enhances aldosterone output, and eliminates the trypsin releasable 18-OH-DOC pool in IEF gels.The data suggest that 18-OH-DOC is synthesised and sequestered in inner zone cells, in a novel non-solvent extractable manner, but can be mobilised for utilisation as an aldosterone precursor in the glomerulosa.