In recent publications from this laboratory we have reported that actinomycin monolactone is formed from actinomycin by an enzyme from Actinoplanes missouriensis1 and as a 'natural' metabolite in the biosynthesis of actinomycin by Streptomyces antibioticus.2 The identification of the monolactone in these instances was made by paper chromatographic and paper ionophoretic comparison with actinomycin monolactone produced by alkaline hydrolysis of actinomycin3 and we were unable to distinguish which of the lactone rings of actinomycin (Fig. 1) had been cleaved and which was intact. During their work on the structure of actinomycin Bullock and Johnson5 oxidized actinomycin with alkaline hydrogen peroxide and obtained in about 2% yield a benzoxazolone-4-carboxylic acid-pentapeptide from ring A of actinomycin D. Unfortunately, under the conditions used, the pentapeptide lactone ring was hydrolyzed and the benzoxazolone carboxylic acid was a rearranged product originating from rings A and B of the actinomycin. Brockmann and Boldt6 overcame the obvious disadvantage of the alkaline conditions by using hydrogen peroxide in acetic acid, but were unable to isolate in pure form the degradation products originating from rings A and B, and were only able to isolate the pentapeptide lactone attached to oxalic acid (from ring C). We concluded that use of ozone might avoid the disadvantage of the alkaline hydrolysis and if the reaction was carefully controlled, the pentapeptide lactone ring would not be destroyed. In a model experiment we ozonized actinocinyl-bis-glycine-methylester and obtained in 2% yield a compound which proved to be 2-oxo-(2H)-3-methoxy-8-methyl-(benzo[1,4]oxazine)-5-carbonyl-glycine methyl ester (Fig. 2). Though this method would be suitable for identification of the two pentapeptide lactone rings of the actinomycins, unfortunately the very low yield does not make it a very practical approach to be applied on a milligram scale to the metabolites available from the fermentations.