Polyene macrolides nystatin and amphotericin B are widely used in the treatment of fungal infections. In order to characterize factors affecting polyene activity, we have isolated Saccharomyces cerevisiae mutants showing selective resistance to nystatin and amphotericin B. Characterization of two of these mutants (nystatin-resistant mutant X1/16 and amphotericin B-resistant mutant X3/33) is presented. Genetic analysis revealed that resistance in each of these mutants is caused by a mutation in one gene with a different mode of inheritance. Nystatin resistance in mutant X1/16 is caused by changes in sterol spectrum while amphotericin B resistance in mutant X3/33 is probably related to modification of the cell wall. Our results suggest that, in spite of their structural similarity, nystatin and amphotericin B differ significantly in mechanisms of their antifungal activity.