Complex, ill-defined mixtures of natural origin are often used as nutrients in the production of biological products through microbial fermentation. Product yields are affected by variation in these natural products. Yeast extract is a typical example of these natural products. Since it is a mixture of amino acids, peptides and nucleic acids, its composition is not well characterized. In this study, we investigated the properties of thiamine hydrochloride, riboflavin and pyridoxine hydrochlride in yeast extract by using a gel filtration chromatography, ion exchange chromatography and high performance liquid chromatography. Yeast extract solution was fractionated by gel filtration chromatography and ion exchange chromatography, and then, each fraction was analyzed by using a high performance liquid chromatography.