A number of cross-linkers that are commonly used in polyacrylamide gels have been incubated with bovine beta-lactoglobulin B and the resulting reaction mixtures were examined by matrix assisted laser desorption/ionization-mass spectrometry. At concentrations of 0.1, 1, and 20 mM of each cross-linker incubated for 1 h with 50 pmol/microL of the protein, a reactivity scale can be expressed as polyethylene glycol diacrylate > N,N'-bisacrylylcystamine > bisacrylyl piperazine > N,N'-methylenebisacrylamide >> N,N'-diallyltartardiamide (PEGDA>BAC>BAP>Bis>>DATD). Relatively short incubation times indicated one of the five Cys residues as the target of reaction, which was confirmed by post-source decay measurements. Longer incubation times (24 h) with bisacrylamide extended the reaction to all five Cys residues and a number of Lys residues. A second consequence of longer reaction time is the involvement of both terminals of the cross-linker in the observed reaction. This experimental evidence is the first to demonstrate a different reactivity of both ends of one of the most commonly used cross-linkers. Investigation of solutions containing a cross-linker and acrylamide monomers provided useful information on the competition between the two identities for reaction with the protein. Possible implications of these experimental observations for isoelectric focusing separations in polyacrylamide gels are discussed.