The basidiomycete Coprinopsis cinerea (Coprinus cinereus) expresses two fruiting body-specific isolectins (CGL1 and CGL2) that belong to the family of galectins. Understanding the role of these beta-galactoside binding lectins is still in the beginning. Even though the prerequisites for substrate binding are well understood, it is not known how discrimination between potential substrates is achieved and what kind of influence this has on the function in a distinct cellular context. Precise knowledge of the expression of galectins and their ligands will aid in elucidating their function. In Coprinopsis, the developmentally regulated ligands for galectins co-localise with galectin expression in the veil surrounding the developing primordium and the outer cells of the young stipe. In addition, galectin ligands are observed in the hymenium. The subcellular localisation of the galectin ligands suggests these to be present in cellular compartments distinct from galectin transport. The sensitivity of the in situ interactions with exogenous galectin towards detergents and organic solvents infers that these ligands are lipid-borne. Accordingly, lipid fractions from primordia are shown to contain galectin-binding compounds. Based on these results and the determined binding specificity towards substituted beta-galactosides we hypothesise that beta-galactoside-containing lipids (basidiolipids) found in mushrooms are physiological ligands for the galectins in C. cinerea.