AbstractFlusilazole is a potent inhibitor ofUstilago maydissporidial growth (I50= 20 μg liter−1). Incorporation of[14C]acetate into ergosterol of growing sporidia is inhibited 50% by 0.5 μg liter−1of the fungicide. Inhibition of ergosterol biosynthesis is concomitant with the accumulation of the precursors eburicol, obtusifoliol and 14α‐methylfecosterol. A novel cell‐free assay has been developed to measure the 14α‐demethylation of[3H]dihydrolanosterol. Flusilazole inhibits the cell‐free demethylation with an I50of 15 μg liter−1.These data provide strong evidence that the mode of action of flusilazole is by inhibiting ergosterol biosynthesis through direct inhibition of the 14α‐demethylation of