The effects of temperature on the three-dimensional organization and on the secondary structure of GPI-anchored 5'-nucleotidase from bull seminal plasma and of its anchor-less form (solubilized ecto-5'-nucleotidase), obtained after GPI anchor removal by phosphatidylinositol-specific phospholipase C were investigated in parallel by circular dichroism and fluorescence spectroscopy. The structural features of the two enzymes were correlated to their functional properties in the temperature range of 25-90 degrees C. The kinetic data indicated that the enzyme activities were temperature dependent, showing the maximal values at 60 degrees C. The relevant Arrhenius plots were linear in the temperature range of 20-60 degrees C and the activation energies were 44.4 and 51.8 kJ/mol for the solubilized and GPI-anchored 5'-nucleotidase, respectively. The time-course measurements of enzyme activity, in the temperature range of 25-55 degrees C, revealed that the two enzymes were of different thermal stability, the solubilized ectoenzyme showing lower thermal deactivation constants and longer half lives. Fluorescence and near UV circular dichroism spectroscopy showed that temperature increases induced remarkable changes in the protein tertiary structure of the two enzymes, whereas far-UV circular dichroism analysis revealed only a small temperature effect on the protein secondary structure content.