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Mutants of Aspergillus fennelliae (Neosartorya fennelliae) resistant to relatively high levels of amphotericin B and low levels of nystatin were obtained by successive transfers of wild type in the presence of increasing concentrations of the polyenes. The resistance of the mutants to the polyenes was accompanied by both qualitative and quantitative changes in the sterol composition of the cells. Those resistant to amphotericin B (AF5-AB1 and p-AB1) lacked ergosterol, the major sterol of the wild type, but contained a new sterol clearly distinguished by the pattern of ultraviolet spectrophotometry and thin-layer chromatography. The mutants resistant to nystatin, however, contained both ergosterol and a new sterol, but the former was produced in a much reduced amount, as compared with the wild type. Genetic analysis indicated that the lack of ergosterol is closely associated with a reduced growth rate, poor asexual reproduction, and the loss of sexual reproduction. Growth studies revealed that the addition of ergosterol to the media did not affect the growth pattern of the mutants. Mutants resistant to amphotericin B showed an increased minimal inhibitory concentration for nystatin, pimaricin, and filipin. Mutants resistant to nystatin, however, conferred increased minimal inhibitory concentration for pimaricin and filipin but not for amphotericin B.