Methanol is one of the most common short‐chain alcohols in fermenting fruits, the natural food of the fruit fly,Drosophila melanogaster. The larvae cope continuously with methanol at various concentrations in order to survive and develop. In the present article, we found toxicities of dietary methanol and formaldehyde were enhanced by piperonyl butoxide, but not by 3‐amino‐1, 2, 4‐triazole, 4‐methylpyrazole, diethylmeleate, and triphenyl phosphate, when assessing by the combination index method. These results reveal that cytochromeP450 monooxygenases (CYPs), rather than catalases, alcohol dehydrogenases, glutathioneS‐transferases, and esterases, participate in methanol metabolism. Moreover, methanol exposure dramatically increasedCYPactivity. The ratios of theCYPactivities in treated larvae to those in control reached, respectively, up to 3.0‐, 3.9‐, and 2.7‐fold, at methanol concentrations of 22.6, 27.9, and 34.5 mg/g diet. In addition, methanol exposure greatly up‐regulated the mRNAexpression level of fiveCyp genes, which wereCyp304a1,Cyp9f2,Cyp28a5,Cyp4d2, andCyp4e2. Their resulting proteins were suggested as the candidate enzymes for methanol metabolism inD. melanogaster larvae. © 2012 Wiley Periodicals, Inc.