Following nitrous acid mutagenesis, one nystatin- (nyl) and two amphotericin B (AB)-resistant (ab1 and ab2) mutants of Candida albicans were isolated and characterized. The three mutants plus a previously described cytochrome P450-deficient mutant (D10) of this organism were analyzed for polyene cross resistance. Cross resistance was noted for ny1 and D10 but not for ab1 and ab2. Sterol analysis indicated that ny1 was a delta 8-delta 7 isomerase mutant while ab1 and ab2 showed wild type sterol profiles. Fatty acid analysis showed no significant differences for ab1, ab2, and ny1 compared to wild type while D10 showed more pronounced differences. AB- and Triton X-100-induced potassium leakage studies indicated that ab1 and ab2 are resistant to low AB levels and ny1 is resistant to higher AB levels. In contrast, ab1 and ab2 were more resistant to detergent-induced potassium leakage than the wild type or mutants ny1 and D10. Significant differences in growth rate, ethanol sensitivity, and response to Tergitol were also noted among the resistant strains. The data indicate a different mechanism of action for the two polyenes and indicate a resistance mechanism for ab1 and ab2 based on subtle alterations of membrane structure rather than sterol substitution.