Pantetheine hydrolase from pig kidney shows a very high resistance to denaturation with chemical denaturants, being unfolded at concentrations of guanidinium chloride higher than 6.5 M. On the contrary, chemical inactivation, followed by recording catalytic activity, occurs before conformational changes can be detected by fluorimetric or spectroscopic measurements. The enzyme resists temperatures as high as 80 degrees C, as monitored by second derivative spectroscopy and circular dichroism. Activity increases with temperature to an optimum of about 70 degrees C recording the initial velocity. The enzyme behaves very differently against chemical denaturants or against temperature denaturation. These results are unusual for a mesophilic protein.