AbstractScreening for resistance to fenpropimorph was undertaken in order to isolate yeast mutants affected in the regulation of the ergosterol pathway. Among the mutants isolated, one bearing the recessivefen1‐1 mutation was characterized by a 1.5‐fold increase in the ergosterol level and a general resistance to sterol biosynthesis inhibitors. Thefen1‐1 mutation was linked toMAT locus on chromosome III. The measurement of enzyme activities involved in the ergosterol pathway revealed that isopentenyl diphosphate (IPP) isomerase activity was specifically increased 1.5‐fold as compared to the wild type strain. However, overexpression of IPP isomerase in the wild type strain was not by itself sufficient to lead to sterol increase or resistance to sterol biosynthesis inhibitors, showing that IPP isomerase is not a limiting step in the pathway. Thefen1‐1 mutation permits viability in aerobiosis of yeast disrupted for sterol‐14 reductase in absence of exogenous ergosterol supplementation, whereas the corresponding strain bearing the wild typeFEN1 allele grows only in anaerobiosis. This result shows that ignosterol is able to efficiently replace ergosterol as bulk membrane component and that thefen1‐1 mutation eliminates the specific ergosterol requirement in yeast.