A process is disclosed for the separation of 4-androsten-3,17-dione/1,4-androstadien-3,17-dione mixtures, which are produced, e.g., upon microbiological sterol-side chain decomposition, through conversion of said mixture into a mixture of 17.beta.-cyano-17.alpha.-hydroxy-4-androsten-3-one/17.beta.-cyano-17.alpha.-hydroxy-1,4-androstadien-3-one, from which the difficultly soluble 17.beta.-cyano-17.alpha.-hydroxy-4-androsten-3-one is separated in crystalline form through filtration, and the 17.beta.-cyano-17.alpha.-hydroxy-1,4-androstadiene remaining in the mother liquor is re-split, directly or after extraction with organic solvent, by treatment in alkaline medium, into 1,4-androstadien-3,17-dione, and isolated as such. The separation of the products follows in high yields, and in sufficient purity for further working-up in the synthesis of androstane and pregnane derivatives, as well as estratrienes.