The adrenal cortex is an architecturally complex tissue, with cellular zonation thought to determine steroidogenesis. The impact that disruption of this tissue's architecture has on steroidogenesis in vitro, particularly adrenal androgen (AA) production, is unclear. We hypothesized that the extent of architectural disruption during tissue preparation would impact the study results. To test this hypothesis, we compared adrenocortical steroidogenesis in freshly prepared tissue slices, minces, and cell suspensions. Normal human adrenals (n = 5, three males and two females, age range 17-43 yr) were obtained at the time of organ donation. The three adrenal tissue preparations were incubated in serum-free medium with 10 μM pregnenolone substrate ± 1 μM ACTH. The production of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, and cortisol in the media were measured by radioimmunoassay. Initial time course incubations using adrenals from a single donor generally demonstrated that minces and suspensions had a greater steroid production compared with slices. In another series of 6-hr incubations using adrenals from four donors, production of dehydroepiandrosterone sulfate was found to be quite sensitive to architectural disruption, i.e. slices less than minces less than suspensions (0.88 vs. 2.1 vs. 3.0 μg/gm tissue, respectively, P < 0.0001). Alternatively, cortisol and androstenedione production was higher in minces compared with slices or suspensions (25.6 vs. 37.7 vs. 18.7 ng/gm tissue, P < 0.0028, and 254 vs. 709 vs. 456 ng/gm tissue, P < 0.0042, respectively). Production of dehydroepiandrosterone was apparently not significantly affected by the type of tissue preparation (28.2 vs. 22.2 vs. 31.2 ng/gm tissue, P < 0.297, respectively). It is unlikely that generalized tissue disruption alone accounted for the observed differences, as the trends among tissue preparations were not consistent among steroids. We conclude that the type of tissue preparation of fresh adrenal tissue impacts significantly on steroidogenesis in vitro.