ABSTRACT Methanol is considered an interesting carbon source in “bio-based” microbial production processes. Since Corynebacterium glutamicum is an important host in industrial biotechnology, in particular for amino acid production, we performed studies of the response of this organism to methanol. The C. glutamicum wild type was able to convert ¹³ C-labeled methanol to ¹³ CO ₂ . Analysis of global gene expression in the presence of methanol revealed several genes of ethanol catabolism to be upregulated, indicating that some of the corresponding enzymes are involved in methanol oxidation. Indeed, a mutant lacking the alcohol dehydrogenase gene adhA showed a 62% reduced methanol consumption rate, indicating that AdhA is mainly responsible for methanol oxidation to formaldehyde. Further studies revealed that oxidation of formaldehyde to formate is catalyzed predominantly by two enzymes, the acetaldehyde dehydrogenase Ald and the mycothiol-dependent formaldehyde dehydrogenase AdhE. The Δ ald Δ adhE and Δ ald Δ mshC deletion mutants were severely impaired in their ability to oxidize formaldehyde, but residual methanol oxidation to CO ₂ was still possible. The oxidation of formate to CO ₂ is catalyzed by the formate dehydrogenase FdhF, recently identified by us. Similar to the case with ethanol, methanol catabolism is subject to carbon catabolite repression in the presence of glucose and is dependent on the transcriptional regulator RamA, which was previously shown to be essential for expression of adhA and ald . In conclusion, we were able to show that C. glutamicum possesses an endogenous pathway for methanol oxidation to CO ₂ and to identify the enzymes and a transcriptional regulator involved in this pathway.