A procedure has been developed, based on ion-exchange chromatography, that readily allows the separation of eleven apparently homogeneous chromium-containing fractions from a brewer's yeast extract. Four of the fractions are amphoteric and show no glucose tolerance factor (GTF) activity, three are classified as negative (two of which are biologically inactive, while the third one shows a slight degree of GTF activity), whereas the four cationic chromium-containing fractions all show varying degrees of GTF activity. Application of the separation procedure to other biological sources of GTF activity resulted in a spectrum of cationic fractions, over the pH range 1.75 to 12, which suggests that GTF cannot be a single species. The cationic chromium-containing fraction from pork kidney powder and fraction P-3 from yeast appear to contain the most GTF-active material and P-3 shows saturation kinetics as expected for a biologically significant substance.