The unfolding and refolding of apohorseradish peroxidase, as a function of guanidinium chloride concentration, were monitored by the intrinsic fluorescence intensity, polarization, and lifetime of the single tryptophan residue. The unfolding was reversible and characterized by at least three distinct stages-the intensity and lifetime data, for example, were both characterized by an initial increase followed by a decrease and then a plateau region. The lifetime data, in the absence and presence of guanidinium chloride, were heterogeneous and fit best to a model consisting of a major Gaussian distribution component and a minor, short discrete component. The observed increase in intensity in the initial stage of the unfolding process is attributed to the conversion of this short component into the longer, distributed component as the guanidinium chloride concentration increases. Our results clarify and amplify previous studies on the unfolding of apohorseradish peroxidase by guanidinium chloride.