Adrenocorticosteroids are known to be synthesized from cholesterol which may arise from de novo synthesis or from the uptake of low-density lipoproteins (LDL) or high-density lipoproteins (HDL). LDL is reported to be a main substrate for corticosteroid synthesis by bovine adrenocortical cells, although the role of HDL, which is well known to be used for steroid biosynthesis in rat adrenals, is still obscure. Therefore, we examined the role of HDL in the regulation of corticosteroidogenesis in bovine adrenals in order to clarify whether or not HDL was selectively utilized for corticosteroid synthesis in vitro. The present data demonstrated that HDL and LDL increased cortisol production in a dose-dependent manner in bovine adrenocortical cells in vitro, and also that HDL cholesterol increased cortisol production significantly higher than LDL cholesterol did. Addition of adrenocorticotrophic hormone (ACTH) with HDL to the incubation media enhanced much higher cortisol production than that with LDL in short time incubation. The present data also demonstrated that uptake of 125I-HDL was significantly greater than that of 125I-LDL. Thus, HDL rather than LDL is thought to be the preferred lipoprotein as a source of steroidogenic substrate cholesterol in bovine adrenal fasciculo-reticularis cells.