An increasing body of evidence suggests that an endogenous mammalian bufadienolide (BD) may be involved in the regulation of Na(+),K(+)-ATPase activity and the pathogenesis of arterial hypertension. We developed a purification scheme for marinobufagenin (MBG), an amphibian cardiotonic BD, and applied it to purify and characterize material in human plasma, culture medium conditioned by Y-1 adrenocortical cells, and rat adrenal tissue. MBG immunoreactivity purified from plasma and measured by ELISA showed important similarities (chromatography and antibody cross-reactivity) to material secreted into cell culture medium by Y-1 cells. This observation indicates that circulating mammalian BD may have an adrenocortical origin. Release of mammalian BD from adrenocortical cells grown in the absence of exogenous cholesterol was reduced by treatment of cultures with mevastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. Supplementation of the serum and cholesterol-free cell culture medium with the LDL fraction of human plasma increased the production of MBG material in the presence of mevastatin, supporting its origin from cholesterol. We used Y-1 cell lines transfected with genes shown to inhibit steroidogenesis through cholesterol side-chain cleavage (Y-1/DAX and Y-1/RIAB) to investigate the dependence of MBG biosynthesis on side-chain cleavage. Our results indicate that the mammalian BD is synthesized in the adrenal cortex from cholesterol and shares important similarities with the amphibian BD MBG, that its biosynthesis is independent of transfer of cholesterol to the side-chain cleavage enzyme complex mediated by steroidogenic acute regulatory protein, and that neither cAMP nor protein kinase A appears to be a critical component of the pathway controlling its biosynthesis.