AbstractThe effect of the four triadimenol stereoisomers on the purified yeast lanosterol 14α‐demethylase (cytochromeP‐45014DM), the primary target of azole antifungal agents, was studied. (1S,2R)‐Triadimenol was the most potent demethylase inhibitor and bound quantitatively to the enzyme below 0.05 μM. This isomer also interfered with the chemical reduction of cytochromeP‐45014DMand the binding of CO to the cytochrome. The other isomers showed a lower inhibitory effect on the enzyme, and the order of activity was (1R,2R)>(1R,2S) ≧ (1S,2S). Based on these findings and the reported preferred conformations for the triadimenol stereoisomers (Anderson, N.H. et al., Pestic. Sci. 15:310–316, 1984), it is predicted that orientation of the hydrophobictert‐butyl andp‐chlorophenyl groups relative to the azole nitrogen is important to fit the antifungal agent in the active site o