Tobacco mosaic virus (TMV) is a rod-shaped virus with 2130 identical protein subunits (17.5 kDa) protecting a 6.4 kb RNA molecule. In this work we compare thermodynamic parameters of the denaturation of TMV by urea and guanidine hydrochloride (GnHCl) combined with high pressure. The viral solution (0.25 mg/mL) in 100 mM Triz HCl and pH 7.0 was incubated at 5ºC in different urea concentrations (0 to 7 M) and guanidina hydrochloride (0 to 4 M), and the pressure effect (up to 220 MPa) on denaturation was followed by spectroscopic measurements. The denaturation degree was estimated by the red shift of center of mass of fluorescence emission spectra. At subdesnaturant concentrations of GnHCl (0.5 to 1.5M) the TMV aggregates, while for urea at subdenaturating concentration (1 to 3M) we observed viral dissociation. Analysis by electron microscopy corroborate these data. The TMV denaturation occurred at GnHCl 3.0 to 4.0 M, or urea 6.0 to 7.0 M at atmospheric pressure. Using high pressure the denaturation was reached with lower concentrations for urea and GnHCl, respectively 2.7 and 1,6 M. The returning to atmospheric pressure have not recovered the spectral changes with pressure of viral samples, indicating a stabilization of denaturated states. The apparent molecular mass of denaturated TMV was smaller for GnHCl experiment than urea experiment, according with HPLC gel filtration results. The variation of Gibbs energy free from denaturation by urea (2.7 to 7M) was 11,06 KJ/mol of subunit of TMV, while for GnHCl (1.6 to 4M) was 4.08 KJ/mol of subunit of TMV. This data shows a higher efficiency of GnHCl in process of denaturation. The study of the thermodynamic parameters in this system can contribute to the understanding of the denaturation mechanisms involved in this process and of the forces that maintain the protein stability.