Production of corticosteroids from the adrenal gland is a multi-step process in which corticosterone is enzymatically processed from its precursor cholesterol. The main hormone regulating the production of corticosterone is the pro-opiomelanocortin (POMC)-derived adrenocorticotropin (ACTH). Adrenals of POMC-deficient (POMC^-/-) mice do not produce corticosterone, neither at basal levels nor in response to acute stimulation with ACTH. However, pharmacological amounts of ACTH delivered continuously elicit corticosterone production over time. To define the relative effects of ACTH on individual factors involved in corticosterone production, parameters of adrenal cholesterol metabolism and steroidogenesis were examined in POMC^-/- mice in comparison to wildtype and ACTH-treated mutant mice. POMC^-/- adrenals lack cholesterol esters (CE); adrenal CE is restored with ACTH treatment. However, discontinuation of ACTH treatment stops corticosterone production despite presence of adrenal CE. Failure of corticosterone production by POMC^-/- adrenals occurs despite the constitutive presence of transcripts of genes required for cholesterol metabolism and steroidogenesis. Levels of key proteins involved in selective-cholesterol uptake and steroidogenesis were attenuated; ACTH treatment increased these protein levels, most significantly those of the receptor responsible for selective uptake of CE, scavenger receptor class B, type I (SR-BI). Our studies reveal that failure of corticosterone production of POMC^-/- adrenal glands and its pharmacological reconstitution by ACTH are not mediated by any one individual protein, but rather as an integrated effect on multiple factors from import of the substrate cholesterol to its conversion to corticosterone.