Isolated adrenal cell preparation was used to investigate the relationship between ascorbic acid and steroidogenesis by two methods: (1) in vivo incorporation of exogenous [1-14C]ascorbic acid into endogenous ascorbic acid of adrenal by intraperitoneal injection of labeled ascorbic acid into rats and studying the depletion of labeled ascorbic acid under a variety of experimental conditions; and (2) study of the uptake of [14C]ascorbic acid by IAC in response to steroidogenic stimuli and various steroids. These studies demonstrate that: 1. IAC preparation by the trypsin digestion method results in almost total depletion of ascorbic acid from adrenal cells, i.e., ascorbic acid content of the cell preparation was less than 1% of the original ascorbic acid in quartered adrenal gland. 2. In spite of such a severe depletion of ascorbic acid, steroidogenesis in response to ACTH and dibutyryl cyclic AMP (dcAMP) is quite pronounced. 3. ACTH and dcAMP affect depletion of endogenously labeled ascorbic acid in IAC by a process that is both concentration- and time-dependent, but is independent of steroidogenic processes. 4. ACTH and dcAMP both inhibit the uptake of exogenous [1-14C]ascorbic acid, which is time-dependent but independent of the steroidogenic phenomenon. 5. The uptake of [14C]ascorbic acid by IAC is independent of extra-to-intracellular gradient of glucocorticoids or mineralocorticoids.