We present here the results of our studies on the ultraviolet rotatory characteristics of β-lactoglobulin in the native as well as in the heat- and guanidine-hydrochloride-denatured states. In comparison to the native protein, the ellipticity at 220 nm, [θ]220, is more negative for the heat-denatured state but more positive for the guanidine-hydrochloride-denatured state. On the basis of other evidence, the heat-denatured state is known to have less ordered structure than the native protein but not as completely disordered as the guanidine-hydrochloride-denatured state. Taking this into account, the observed changes in [θ]220 on denaturation have been interpreted in terms of anomalous effects that could arise from the exposure, during denaturation, of asymmetrically-placed aromatic residues or of β structure regions situated in a hydrophobic environment in the native protein molecule. During the transition from the native to the guanidine-hydrochloride-denatured state, [θ]220 is found to go through a change towards more negative values at lower concentrations of guanidine hydrochloride (< 2.3 M) before changing to more positive values at higher concentrations of the denaturant. This, in conjunction with the noncoincidence of the transition profiles obtained by [θ]220 on the one hand and the ellipticity or difference absorbance at 290 nm on the other, provides evidence for the presence of an intermediate state in the early stages of the denaturation of β-lactoglobulin by guanidine hydrochloride.