Mutants of Ustilago maydis (DC) Corda with high resistance to azoxystrobin (RF 164 to 4714, based on EC50 values), an inhibitor of mitochondrial electron transport at the cytochrome bc1 complex, were isolated in a mutation frequency of 2.3 x 10(-7) after nitrosoguanidine mutagenesis and selection on media containing 1 microgram ml-1 azoxystrobin in addition to 0.5 mM salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strains by azoxystrobin (1.5 micrograms ml-1) in addition to SHAM (1 mM), but not in the mutant isolates. Genetic analysis with nine such mutant isolates resulted in progeny phenotypes which did not follow Mendelian segregation, but satisfied the criteria of non-Mendelian (cytoplasmic) heredity. In crosses between three mutant isolates with the compatible wild-type strains, the sensitivity was inherited by progeny maternally from the wild-type parent strain (criterion of uniparental inheritance). In crosses between wild-type strains and remaining mutant isolates, a continuous distribution of sensitivity in the progeny was found (criterion of vegetative segregation). The third criterion of cytoplasmic resistance (criterion of intracellular selection) was fulfilled by experiments on the stability of resistance phenotypes. With two exceptions, a reduction of resistance was observed in the mutant strains when they were grown on inhibitor-free medium. Recovery of the high resistance level was observed after they were returned to the selection medium. Cross-resistance studies with other fungicides, which also inhibit electron transport through complex III of respiratory chain, showed that mutations for resistance to azoxystrobin were also responsible for reduced sensitivity to kresoxim-methyl (RF 18 to 1199) and to antimycin-A (RF 20 to 305), which act at the Qo and Qi sites of the cytochrome bc1 complex, respectively. Studies of the fitness of azoxystrobin-resistant isolates showed that these mutations appeared to be pleiotropic, having significant adverse effects on growth in liquid culture and pathogenicity on young corn plants.