Oligomerization directs active site formation in homotrimeric 2'-deoxyuridine triphosphate pyrophosphatases (dUTPases). Stability of the homotrimer is a central determinant in enzyme function. The present comparative studies of bacterial and fruitfly dUTPases with homologous 3D structures by differential scanning microcalorimetry; fluorescence, circular dichorism and infrared spectroscopies, demonstrate that unfolding is a two-state highly cooperative transition in both dUTPases excluding a significantly populated intermediate state of dissociated and folded monomers. The eukaryotic protein is much less resistant against either thermal or guanidine hydrochloride-induced denaturation. Results suggest that hydrophobic packing of the inner threefold channel of the dUTPase homotrimer greatly contributes to stability.