Alternative oxidase (AOX) is dramatically induced when the fungus Magnaporthe grisea is incubated with the fungicide SSF-126, which interacts with the cytochrome bc1 complex in the electron transport system of mitochondria. A full-length cDNA for the alternative oxidase gene (AOX) was obtained, and the deduced amino acid sequence revealed marked similarity to other AOXs, but lacks two cysteine residues at corresponding sites which are conserved in plant AOXs and play essential roles in the post-translational regulation. Northern blot experiments showed that treatment of M. grisea cells with SSF-126 induces accumulation of AOX mRNA in a dose-dependent manner, and the level was correlated with the activity of alternative respiration. H2O2 also induced the accumulation of the transcript with a short half-life (<15 min). Nuclear run-on experiments showed that the AOX gene was transcribed constitutively in unstimulated cells. Cycloheximide did not change the basal level of transcription, but induced the accumulation of the transcript, indicating that active degradation of the transcript occurs by factor(s) sensitive to cycloheximide. On the other hand, SSF-126 enhanced the transcriptional activity of AOX gene threefold compared to that of control cells, and H2O2 was also potent for enhancement of the transcription. From these results, it is concluded that the respiratory inhibitor-dependent activation of the transcription is a primary determinant for the induction of alternative respiration in M. grisea. Because we have previously shown that SSF-126 treatment of M. grisea mitochondria induced the generation of superoxide, active oxygen species are thought to be signal mediators to activate AOX gene transcription in M. grisea.