Azole antifungal compounds inhibit sterol 14 alpha-demethylase. They are used extensively for the treatment of immunocompromised patients where fungal infection is common and often results in death. Resistance to the compounds is emerging, particularly in fungal pathogens obtained from AIDS patients undergoing prolonged therapy. We show here that cell growth arrest correlates with the accumulation of 14 alpha-methyl-ergosta-8,24(28)-dien-3 beta,6 alpha-diol in a yeast strain with a sterol 14 alpha-demethylase gene disruption, which mimics stringent treatment conditions. Cells can overcome the effect of such a block by a suppressor mutation in sterol delta 5,6 desaturation and acquire azole resistance. Plasmid-based complementation of sterol 14 alpha-demethylase defect does not alter the azole susceptibility of strains containing these suppressor mutations, showing resistance is due entirely to the delta 5.6 desaturase defect.