We have employed a combination of cysteine mutagenesis and chemical crosslinking using a photoactivatable sulfhydryl reagent, benzophenone-4-maleimide, to obtain a covalent complex between human galectin-1 and a model glycoprotein ligand, asialofetuin. We previously obtained a crosslinked product when Lys²⁸ of the cysteine-less form of human galectin-1 was mutated to cysteine. To investigate whether substituting either of the two flanking amino acid residues in the same β-strand, Ala²⁷ and Ser²⁹, to cysteine could result in crosslinking to the bound asialofetuin, two cysteine-containing mutants were generated. Although both the mutants adsorbed to asialofetuin-agarose and were eluted with 0.1 M lactose, confirming their ability to interact with asialofetuin, these mutants did not crosslink to the bound glycoprotein ligand following treatment with benzophenone-4-maleimide. Therefore the orientation of the side chain of the introduced cysteine residue apparently plays an important role in the crosslinking reaction.