An enzyme responsible for inactivating the peptidolactoneantibiotic etamycin was partially purified fromextracts of Streptomyces lividans. On-line liquidchromatography−electrospray mass spectrometry showed thattheproduct of the enzyme-catalyzed reaction had a different retention timebut the same mass as etamycin. Reaction ofetamycin in 1.0 M NaOH gave a mixture consisting of the productexpected from hydrolysis of the lactone bond anda product corresponding to that from the enzyme reaction. Labelfrom H218O was incorporated into thehydrolysisproduct but not into the product of the enzyme reaction. Anenzyme-catalyzed elimination reaction is proposed, andis supported by evidence for the structure of the resultingdehydrobutyrine peptide from combined liquidchromatography−tandem mass spectrometry (LC−MS−MS) and1H NMR spectroscopy.