We analyzed the pathogenicity of chitin synthetase (chs) disruptants of Ustilago maydis obtained with the carboxin-resistant or the hygromycin-resistant cassettes. We found that only chitin synthetase (chs) mutants obtained by gene disruption with:the carboxin resistance cassette lost their virulence to maize (Zea mays) seedlings. Carboxin is a systemic fungicide that inhibits respiration by preventing the oxidation of succinate. We demonstrated that carboxin-resistant transformants were affected in the levels of succinate dehydrogenase and respiratory activities when compared with hygromycin-resistant disruptants. We propose that loss of virulence in the carboxin-resistant transformants is owing to loss of respiratory fitness, which probably represents an important component of virulence in this fungus.