Mycobacterial glycopeptidolipid (GPL) interactions with membranes were analysed with monolayer experiment, using GPLs bearing 3, 1, or 0 carbohydrate residues (GPL3, GPL1, GPL0). Compression isotherms and surface potential determinations suggested that the glycopeptidic moiety of GPL3 permanently dipped in water, while those of GPL1 and GPL0 can lay in the interface. Insertion of GPL molecules into a preformed phospholipid monolayer was observed using GPL3 or GPL1 dispersions, but not from GPL0. It is postulated that the activity of GPL0 is low due to its failure to become inserted into membranes, as is that of GPL3 owing to its insertion only by its acyl chain. GPL1 is likely to disturb membranes by inserting its glycopeptidic moiety into the interface.