Galectin-3 is a multifunctional carbohydrate-binding protein with roles in cancer progression. Besides carbohydrate-dependent extracellular functions, galectin-3 participates in carbohydrate-independent intracellular signalling pathways, including apoptosis, via protein-protein interactions, some engaging the carbohydrate-binding groove. When ligands bind within this site conformational rearrangements are induced and therefore information of un-liganded galectin-3 is valuable for structure-based drug-design. Removal of co-crystallised lactose from the human galectin-3 carbohydrate-recognition domain was achieved via crystal soaking, but took weeks, despite low affinity. Pre-soaking to remove lactose enabled subsequent binding of cryo-protectant glycerol, whereas when lactose was not removed a priori then the glycerol could not displace it over the short cryo-soak timeframe. Slow diffusion of lactose removal from crystals contrasts glycerol entering within minutes. The importance of removal of incumbent ligands prior to attempts to introduce alternative ligands is indicated, even for proteins exhibiting low affinity for ligands, and has significance for ligand exchange in structure based drug desig