Phosphatidyl-myo-inositol mannosides (PIM) are not only important structural components of the mycobacterial envelope but also are major non-peptidic antigens of the host innate and acquired immune responses. Indeed, they are ligands of TLR-2 and they activate CD1-restricted T lymphocytes. In addition, PIM constitute the basic structure of the lipidic anchor of two lipoglycans, lipomannans and lipoarabinomannans, which are important immunomodulators in the course of tuberculosis. The fatty acyl substituents present on PIM molecules play a crucial role for both their physical properties and biological activities. PIM contain four acylation sites, two on the glycerol, one on a mannose, and one on the myo-inositol units. We propose here an analytical procedure, based on mass spectrometry, to determine the structure of the fatty acids present on each of these different acylation sites. We show that the nature of the fatty acids located on both positions of glycerol can be deduced from IRMPD analysis of negative precursor ions from native PIM species, while the fatty acids located on myo-inositol and mannose units can be identified by MALDI-TOF CID MS of protonated and cationized molecular ions. Thus, the combination of MS/MS data obtained from positive and negative pseudomolecular ions generated by ESI or MALDI appears as a powerful approach for the structural characterization of the PIM acyl form structure.