Resistance to azole antifungals in Ustilago maydis was associated with a leaky defect in sterol Δ5(6) desaturase. This defect resulted in reduced accumulation of 14α-methylergosta-24(28)-diene-3β,6α-diol and an increase in the proportion of 14α-methylfecosterol in treated cells when compared to the parent strain. The results demonstrate the importance of this mechanism in pathogenic fungi.