The aim of this study was to purify the glucose tolerance factor (GFT) from yeast, and to gain further knowledge on its chemical structure. Activity of GTF was determined by its potency to stimulate 1-14C glucose oxidation of rat adipose tissue, in vitro, in the presence of maximally effective insulin concentrations. While described procedures Toepfer, Merth, Polansky, Roginski and Wolf (1977) resulted in some 3-fold purification, specific activity was no more increased by further fractionation on HPLC and/or TLC. Instead separation on TLC yielded several fractions with GTF activity, two of them enriched in aspartate and cystine/cystein, respectively, being the most important. L-aspartate, when added at comparable concentrations in vitro displayed similar GTF-activity as did adenosine, another component present in yeast extracts. It is concluded that GTF-activity of yeasts is not attributable to a single molecular species but rather to the combined action of amino acids and nucleosides especially aspartate, and adenosine.