The biosynthesis of digitalis-like compounds (DLC) was determined in bovine and rat adrenal homogenates, as well as in primary rat adrenal cells, by following changes in the concentration of DLC using three independent sensitive bioassays: inhibition of [3H]-ouabain binding to red blood cells and competitive ouabain and bufalin ELISA. The amounts of DLC in bovine and rat adrenal homogenates, as measured by the two first bioassays, increased with time when the mixtures were incubated under tissue culture conditions. Rat primary adrenal cells were incubated in the presence of [1,2-(3)H]-25-hydroxycholesterol, [26,27-(3)H]-25-hydroxycholesterol or [7-(3)H]-pregnenolone. The radioactive products, as well as the digitalis-like activity, were fractionated by three sequential chromatography systems. When [1,2-(3)H]-25-hydroxycholesterol or [7-(3)H]-pregnenolone was added to the culture medium, the radioactivity was co-eluted with digitalis-like activity, suggesting that at least one of the DLC might originate in hydroxycholesterol. In contrast, when the culture medium was supplemented with [26,27-(3)H]-25-hydroxycholesterol, the radioactivity was not co-eluted with the digitalis-like activity, indicating that side chain cleavage is the first step in the synthesis of digitalis-like compounds by rat adrenal.