We describe the cooperativity in the biotin binding of streptavidin. We have developed an electrophoretic method which can separate streptavidin molecules with bound biotin from those without biotin. In 6 M urea, the electrophoretic mobility of streptavidin in polyacrylamide gels becomes significantly faster upon biotin binding. When streptavidin was titrated with biotin, only two major bands were observed on the gel, consisting of streptavidin molecules without bound biotin and those saturated with biotin. The change in mobility is due partly to the negative charge of the bound biotin, but it must reflect conformational changes of the protein molecule associated with biotin binding. Gel filtration chromatography showed that the streptavidin molecule dissociates into two subunit dimers in the presence of 6 M urea. These results suggest that the biotin binding by the streptavidin subunit dimer is cooperative and that some communication must exist between the two subunits.