Thermal stability of Escherichia coli Fpg protein was studied using far-UV circular dichroism and intrinsic fluorescence. Experimental data indicate that Fpg irreversibly aggregates under heating above 35 degreesC. Heat aggregation is preceded by tertiary conformational changes of Fpg. However, the secondary structure of the fraction that does not aggregate remains unchanged up to similar to 60 degreesC. The kinetics of heat aggregation occurs with an activation enthalpy of similar to 21 kcal/mol. The fraction of monomers forming aggregates decreases with increasing urea concentration, with essentially no aggregation observed above similar to3 M urea, suggesting that heat aggregation results from hydrophobic association of partially unfolded proteins. With increasing urea concentration, Fpg unfolds in a two-state reversible transition, with a stability of similar to3.6 kcal/mol at 25 degreesC. An excellent correlation is observed between the unfolded fraction and loss of activity of Fpg. A simple kinetic scheme that describes both the rates and the extent of aggregation at each temperature is presented.