Abstract Phosphatidylinositol mannosides (PIM) and their multiglycosylated counterparts, lipomannan (LM) and lipoarabinomannan (LAM), are complex glycolipids and lipoglycans found in the cell envelopes of all mycobacterial species. They play various essential although poorly defined roles in mycobacterial physiology and are important immunomodulatory molecules in the course of tuberculosis and leprosy as well as key ligands promoting the entry of mycobacteria and their survival within phagocytic and non-phagocytic cells. Although much progress has been made over the last 25 years in elucidating the structures and biosynthesis of these molecules, fundamental questions remain about the pathways leading to their biosynthesis and translocation to the cell surface. Furthermore, while the pleiotropic biological activities displayed by purified PIM, LM and LAM in cellular models suggest that they play important roles in pathogenesis, studies aimed at validating this assumption and precisely delineating their contribution to host-pathogen interactions when carried by intact bacilli are still limited by the paucity of mutants deficient in well-defined aspects of the biosynthesis and export of these molecules that are available. We propose to pursue structural, genetic and biochemical studies toward a complete definition of the structure (Aim 1), biosynthesis (Aim 2) and export (Aim 3) of PIM, LM and LAM. Completing our understanding of PIM, LM and LAM biogenesis, in addition to providing fundamental knowledge about the biochemistry of Mycobacterium tuberculosis (Mtb), is expected to lead to the discovery of essential enzymes and transporters which, much like the arabinosyltransferases of the Emb family and the epimerase DprE1, could provide new opportunities for anti-tuberculosis drug development. The availability of recombinant strains accumulating structurally defined biosynthetic precursors will facilitate structure-function relationship studies, and that of defined Mtb mutants deficient in various aspects of PIM, LM and LAM synthesis will allow a precise assessment of the contribution of these molecules to the immunopathogenesis of tuberculosis. Abbreviations: AG, arabinogalactan; AM, arabinomannan; AcylT, acyltransferase; Araf, arabinofuranosyl; AraT, arabinosyltransferase; CZE, capillary zone electrophoresis; DOC, deoxycholate; GT, glycosyltransferase; Ino, myo-Inositol; LAM, lipoarabinomannan; LM, lipomannan; LPS, lipopolysaccharide; MALDI-TOF, Matrix-Assisted Laser desorption/ionization time of flight; Manp, mannopyranosyl; ManT, mannosyltransferase; MPI, mannosylated phosphatidylinositol; MS, mass spectrometry; MTX, methyl-thio-xylose; ORF, open reading frame; OM, outer membrane; PIM, phosphatidyl-myo-inositol mannosides; TLC, thin-layer chromatography. Nomenclature: PIM is used to describe the global family of phosphatidylinositol mannosides that carries one to four fatty acids (attached to the glycerol, inositol and/or mannose) and one to six m...