The esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus is a monomeric protein with a molecular weight of about 35.5 kDa. The enzyme is barely active at room temperature, displaying the maximal enzyme activity at about 80Β°C. We have investigated the effect of the temperature on the protein structure by Fourier-transform infrared spectroscopy. The data show that between 20Β°C and 60Β°C a small but significant decrease of the β€-sheet bands occurred, indicating a partial loss of β€-sheets. This finding may be surprising for a thermophilic protein and suggests the presence of a temperaturesensitive β€-sheet. The increase in temperature from 60Β°C to 98Β°C induced a decrease of β£-helix and β€-sheet bands which, however, are still easily detected at 98Β°C indicating that at this temperature some secondary structure elements of the protein remain intact. The conformational dynamics of the esterase were investigated by frequencydomain fluorometry and anisotropy decays. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. Remarkably, the tryptophanyl fluorescence emission reveals that the indolic residues remained shielded from the solvent up to 80Β°C, as shown from the emission spectra and by acrylamide quenching experiments. The relationship between enzyme activity and protein structure is discussed. Proteins 2000;38:351-360.