Human recombinant glycine N-methyltransferase (GNMT) unfolding by urea was studied by enzyme activity, size-exclusion chromatography, fluorescence spectroscopy, and circular dichroism. Urea unfolding of GNMT is a two-step process. The first transition is a reversible dissociation of the GNMT tetramer to compact monomers in 1.0-3.5M urea with enzyme inactivation. The compact monomers were characterized by Stokes radius (R(s)) of 40.7A equal to that of globular proteins with the same molecular mass as GNMT monomers, absence of exposure of tryptophan residues into solvent, and presence of about 50% of secondary structure of native protein. The second step of GNMT unfolding is a reversible transition of monomers from compact to a fully unfolded state with R(s) of 50A, exposed tryptophan residues, and disrupted secondary structure in 8M urea.