A novel acetol monooxygenase has been detected in Mycobacterium Py1. Extracts of 1,2‐propanediol‐grown cells oxidized acetol in an oxygen‐and NADPH‐consuming reaction. The initial oxidation product is probably hydroxymethyleneacetate, which spontaneously rearranges to acetate and formaldehyde. Acetol oxidation was inhibited by carbon monoxide, but not by 10 mM cyanide, indicating that a cytochrome P‐450 type oxygenase might be involved. The enzyme activity was only detected in 1,2‐propanediol‐ or acetol‐grown cells, suggesting that this acetol monooxygenase plays a role in the metabolism of 1,2‐propanediol by Mycobacterium Py1.