A Mycobacterium leprae-specific phenolic glycolipid antigen was purified from Formalin-fixed liver preserved from an advanced lepromatous leprosy patient. Its chemical and immunological properties were compared with those of phenolic glycolipid-I obtained from M. leprae-infected armadillo liver. Based on the findings that the glycolipids from the two sources have the same thin-layer chromatographic properties, infrared absorption spectrum, sugar composition, and seroreactivity, we conclude that large quantities of the phenolic glycolipid-I antigen are produced in human lepromatous leprosy lesions and that Formalin-fixed lepromatous livers and spleens from the prechemotherapeutic era are suitable sources of the glycolipid.