1. 1. Cationic fractions were isolated from a low chromium (< 0.2 ppm) commercial yeast extract in an attempt to purify the material responsible for glucose tolerance factor (GTF) activity observed in a standard yeast assay system. 2. 2. Following previously described procedures a fraction with GTF activity but containing negligible chromium was isolated, which on further purification was found to be composed of many separate small basic peptides. 3. 3. Much of the activity of the yeast GTF material in the yeast assay could be attributed to the presence of basic peptides and free amino acids acting as nitrogen sources for the yeast. 4. 4. Additional activity was present in the yeast GTF sample, which was not due to a synergistic effect of the mixed amino acids and peptides although the component of the yeast extract responsible for this activity was not identified. 5. 5. The results show that the GTF fractions isolated according to most previously published procedures are highly impure, and conclusions drawn about the nature of GTF based on these isolates must remain open to question. 6. 6. The activity due to the presence of peptides and amino acids is a major cause of lack of specificity of the yeast system as an assay for GTF.