Sublethal enrichment was used to generate mutants of Pythium sylvaticum tolerant to kanamycin and tetracycline. Kanamycin tolerance was readily generated, and mutants had growth rates similar to wild-type isolates at antibiotic concentrations lethal to wild-type isolates. Based on crosses between wild-type and mutant isolates, two types of inheritance of tolerance were identified. A high level of kanamycin tolerance was inherited in progeny only when the maternal parent was drug tolerant and was correlated with the inheritance of maternal mitochondrial DNA. A lower level of tolerance was observed in some progeny when the paternal parent was tolerant to the antibiotic and, based on the lack of inheritance of paternal mitochondrial DNA, was presumably nuclear-encoded. Selection of mutants tolerant to tetracycline took longer to generate than kanamycin-tolerant mutants. Based on crosses between tolerant and wild-type parents, tolerance to tetracycline was nuclear-encoded. Tolerance to both antibiotics was stable, with cultures retaining tolerance following repeated transfers on nonamended medium and after storage for 7 years.