Fluorescence and circular dichroism spectroscopy as well as analytical ultracentrifugation and glutaraldehyde cross-linking were utilized to evaluate the tertiary and quaternary structural changes occurring on the denaturation and reconstitution pathways of transthyretin (TTR) as a function of guanidine hydrochloride (GdnHCl) concentration. These results demonstrate that the GdnHCl-mediated denaturation and reconstitution of TTR is reversible. However, the lowest GdnHCl concentration that dissociates and unfolds transthyretin does not allow the unfolded monomer to refold to tetramer at a rate that is measurable. As a result, there is a striking hysteresis observed upon comparison of the GdnHCl-mediated denaturation and reconstitution transitions. The TTR tetramer does not dissociate into unfolded monomer until the denaturant concentration exceeds 4 M GdnHCl, whereas unfolded monomeric TTR (denatured in 7 M GdnHCl) does not refold and assemble into a native tetrameric structure until the GdnHCl concentration is reduced to less than 2 M. These results imply that a significant kinetic barrier intervenes between the folded tetramer and unfolded monomer in both the denaturation and reconstitution directions at pH 7. A kinetics study of the denaturation of TTR as a function of GdnHCl concentration yields a first-order rate constant for unfolding of (9.0 +/- 7.5) x 10(-11) s-1, estimated by extrapolation of the rate constants for the tetramer to unfolded monomer transition as a function of GdnHCl to 0 M GdnHCl. This rate is very slow; as a result, wild-type TTR is predicted to be kinetically stable as a tetrameric quaternary structure once formed. These results imply that the rate of TTR dissociation and partial unfolding to the monomeric amyloidogenic intermediate under denaturing conditions may play a role in transthyretin-based amyloid diseases.