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A novel antitumor antibiotic, methylenolactocin, was isolated from the culture nitrate of a new isolate of fungus identified as Penicillium sp. The fermentation yield reached about 100 mg per liter of the broth. Methylenolactocin has the molecular formula of CnHi6O4 and possess an exomethylene lactone structure. Its structure was determined to be 3-carboxy-2-methylene-4-nonanolide by spectroscopic data. It is active against some Gram-positive bacteria and it prolongs the life span of mice inoculated with Ehrlich carcinoma.