In order to determine the conformational relationship of iron binding of human serotransferrin and lactotransferrin, ultraviolet difference spectral studies were performed in the presence of guanidine chloride and perturbants as deuterium oxide, ethylene glycol, glycerol and polyethylene glycol. In the presence of guanidine chloride solution the molar absorption differences at 292 nm of iron-saturated forms versus iron-free forms of human serotransferrin and lactotransferrin are respectively -16000 +/- 1000 and -14000 +/- 775. These modifications may be attributed to the involvement of tryptophan residues in the iron-binding sites of the two proteins. However, the results do not demonstrate that these tryptophan residues are bound directly to iron. Difference spectral studies in the presence of perturbants show that the apparent exposed tryptophan and tyrosine residues are higher with shorter range perturbants in iron-free forms of both transferrin molecules. The most important modification of exposed tyrosine residues has been noticed upon removing iron from human lactotransferrin than from serotransferrin.