Abstract: Individual cells within isogenic microbial cultures exhibit phenotypic heterogeneity, an issue that is attracting intense interest. Heterogeneity could confer benefits, in generating variant subpopulations that may be better equipped to persist during perturbation. We tested this hypothesis by comparing the survival of wild-type Saccharomyces cerevisiae with that of mutants which are considered stress-sensitive but which, we demonstrate, also have increased heterogeneity. The mutants (e.g. vma3, ctr1, sod1) exhibited the anticipated sensitivities to intermediate doses of nickel, copper, alkaline pH, menadione or paraquat. However, enhanced heterogeneity meant that the resistances of individual mutant cells spanned a broad range, and at high stress occasional-cell survival in most of these populations overtook that of the wild type. Green fluorescent protein (GFP) reporter studies showed that this heterogeneity-dependent advantage was not related to perturbation of buffered gene expression. Deletion strain screens combined with other approaches revealed that vacuolar alkalinization resulting from loss of Vma-dependent vacuolar H(+)-ATPase activity was not the cause of vma mutants' net stress sensitivities. An alternative Vma-dependent resistance mechanism was found to suppress an influence of variable vacuolar pH on the metal resistances of individual wild-type cells. In addition to revealing new mechanisms of heterogeneity generation, the results demonstrate experimentally a benefit under adverse conditions that arises specifically from heterogeneity, and in populations conventionally considered to be disadvantaged.