Activity assays, conformational changes and transitional switches between secondary structures of a peroxidase from Euphorbia characias were studied in the presence of trifluoroethanol and in the presence or absence of calcium ions. The addition of trifluoroethanol up to 10-20% first induced a drastic decrease of alpha-helix content followed by an increase of tryptophan fluorescence emission intensity, a progressive re-induction of the formation of alpha-helical elements concomitant with loss of enzyme activity. In the presence of calcium ions, the fluorescence of the enzyme almost remained unchanged in the trifluoroethanol concentration range 5-20%. Further increase in trifluoroethanol concentration led to a protein structure characterized by a progressive re-induction of alpha-helical elements, a remarkable increase of the tryptophan fluorescence and a loss of enzyme activity. These results indicate that calcium ions in Euphorbia peroxidase play an essential role in maintaining the hydrophobic interactions on the protein structure preserving enzymatic activity.