Triose phosphate isomerase (TIM) was prepared and purified from chicken breast muscle. The equilibrium unfolding of TIM induced by guanidine hydrochloride was investigated by following the changes in intrinsic fluorescence, circular dichroism and second-derivative spectroscopy. Results show that the unfolding of TIM is highly cooperative and no folding intermediate was detected in the experimental conditions used. The thermodynamic parameters of TIM during guanidine denaturation were calculated according to a two-state unimolecular unfolding model. Thermal denaturation of TIM monitored by CD at 222 nm shows also a single, cooperative transition with an apparent T_m of 64.6℃ and the thermal stability of TIM was decreased in the presence of low concentrations of guanidine. The possible unfolding pathway of the dimeric TIM was discussed. It shows that the secondary and tertiary structural changes of TIM occur simultaneously during guanidine denaturation and the unfolding of dimeric TIM follows an apparent two-state transition without detectable dissociation model.