Previous studies have found that, when injected into mice, glycolipidic fractions of mycobacterial cell walls containing phosphatidylinositol mannosides (PIM) induced a granuloma and recruitment of Natural Killer T cells in the lesions. The dimannoside (PIM(2)) and the hexamannoside (PIM(6)) PIM were isolated from Mycobacterium bovis bacillus Calmette Guérin and shown to act alike, but the activity was found to be dependent on the presence of the lipidic part. The chemical structure of PIM was then re-evaluated, focusing on the characterization of their lipidic part, defining mono- to tetra-acylated PIM(2). The structure of these acyl forms was elucidated using a sophisticated combination of chemical degradations and analytical tools including electrospray ionization/mass spectrometry, electrospray ionization/mass spectrometry/mass spectrometry, and two-dimensional NMR. Finally, the acyl forms were purified by hydrophobic interaction chromatography and tested for their capacity to induce the granuloma and Natural Killer T cell recruitment. We found that there is an absolute requirement for the molecules to possess at least one fatty acyl chain, but the number, location, and size of the acyl chains was without effect. Moreover, increasing the complexity of the carbohydrate moiety did not lead to significant differences in the biological responses.