The triazole fungicide etaconazole (CGA64 251) interferes with the ergosterol biosynthesis in Ustilago maydis by inhibiting the C-14 demethylation of the sterol nucleus. During the late log growth phase of U. maydis a novel endogenous sterol metabolite (14α-methyl-ergosta-8,24(28)- dien-3β,6α-diol) was discovered and analyzed, which accumulates under the influence of the fungicide. The structure of this metabolite points to a hydroxylation-dehydration mechanism for the introduction of the double bond at C-5 during the ergosterol biosynthesis.