AbstractBiological macromolecules evolved to perform their function in specific cellular environment (subcellular compartments or tissues); therefore, they should be adapted to the biophysical characteristics of the corresponding environment, one of them being the characteristic pH. Many macromolecular properties are pH dependent, such as activity and stability. However, only activity is biologically important, while stability may not be crucial for the corresponding reaction. Here, we show that the pH‐optimum of activity (the pH of maximal activity) is correlated with the pH‐optimum of stability (the pH of maximal stability) on a set of 310 proteins with available experimental data. We speculate that such a correlation is needed to allow the corresponding macromolecules to tolerate small pH fluctuations that are inevitable with cellular function. Our findings rationalize the efforts of correlating the pH of maximal stability and the characteristic pH of subcellular compartments, as only pH of activity is subject of evolutionary pressure. In addition, our analysis confirmed the previous observation that pH‐optimum of activity and stability are not correlated with the isoelectric point, pI, or with the optimal temperature. Proteins 2010. © 2010 Wiley‐Liss, Inc.