AbstractWhilein vitroincubation of dispersed cell preparations of adrenal cell types has been widely used as an experimental model, few studies have addressed the possibility that the enzymic and mechanical treatments involved may affect tissue functions. Using rat adrenal whole capsule tissue, consisting of glomerulosa cells still attached to the connective tissue capsule together with some fasciculata cells, and dispersed glomerulosa cell preparations formed by a variety of enzymic and incubation treatments, striking differences have been demonstrated between the functions of the various preparations in vitro. Under ACTH stimulation, whole capsules produced (ng per pair ± s.e.) 405 ± 35 ng aldosterone, 650 ± 60 ng 18‐hydroxycorticosterone (18‐OH‐B) and 850 ± 90 ng corticosterone. In cells dispersed by collagenase incubation followed by repeated pipetting and filtration, aldosterone and 18‐OH‐B yields under ACTH stimulation fell to values less than 10% of those produced by whole tissue, whereas corticosterone values were unchanged. Omitting the filtration step gave a less well marked decline in aldosterone and 18‐OH‐B to 50% of intact tissue values. When the tissue was not dispersed after collagenase incubation, aldosterone and 18‐OH‐B outputs were similar in the two preparations. The decline in aldosterone and 18‐OH‐B is not attributable to loss in cell–cell contact alone, since short term culture of collagenase dispersed cells on contracting collagen discs did not restore the capacity to produce these steroids, and a decline in their output also occurred in similar culture of intact capsule tissue. In acute incubations, hyaluronidase had similar effects to collagenase, whereas trypsin, papain and a bacterial protease evoked aldosterone release during the preincubation period, but did not affect subsequent yields of aldosterone and 18‐OH‐B in incubations of dispersed (but not filtered tissue) in the presence of ACTH. Chymo‐trypsin had no effect on preincubation but eliminated subsequent response to ACTH in all incubation conditions. Together with previously published data on the effects of trypsin, the results support the view that in intact rat adrenal glomerulosa tissue, aldosterone and 18‐OH‐B are sequestered into intracellular stores in the form of novel steroid‐protein complexes. These are hydrolysed by trypsin and other preoteases with consequent release of steroid, but are virtually eliminated by conventional methods of cell suspension preparations, using collagenase preincubation with subsequ