In the previous report, it was observed qualitatively that O-benzoylthiamine disulfide (BTDS) and other symmetrical thiamine disulfide were reduced to free thiamine and further some part of them was converted to protein-thiamine complexes by the reaction with denatured egg albumin or serum albumin. In this report, quantitative investigations of complex formation were attempted. The complexes were separated from the reaction mixture by means of metaphosphoric acid deproteinization and the combined thiamine was determined by the thiochrome method, after the liberation of thiamine from the complex by treating with sodium thiosulfate. Then, the reaction of BTDS or its related compounds with the SH group of protein and the effects of pH, temperature and time etc. on the reaction were investigatigated. As a result of the experiment, these effects on the complex formation were made clear and it was confirmed that the complex was protein-thiamine mixed disulfide which was produced by the reaction with the SH groups of protein.