Mycobacterium "habana" strain TMC 5135, which has been proposed as a vaccine against both leprosy and tuberculosis, is considered to be a strain of serotype I of the recognized species Mycobacterium simiae. We have now shown that each of these strains possesses characteristic polar glycopeptidolipids (GPL) which are sufficiently different to allow unequivocal strain identification. Thin layer chromatographic analysis demonstrated that M. habana synthesizes a family of apolar GPLs and three distinct polar GPLs (pGPL-I to -III) which exhibited migration patterns different from those of M. simiae serotype I (pGPL-Sim). Using a combination of chemical, mass spectrometric, and proton-NMR analyses, the GPLs from M. habana were determined to be based on the same generic structure as those from the M.