The ability to predict the absolute stability of proteins based on their corresponding sequence and structure is a problem of great fundamental and practical importance. In this work, we report an extensive, refinement and validation of our recent approach (Roca et al., FEBS Lett 2007;581:2065–2071) for predicting absolute values of protein stability ΔGfold. This approach employs the semimacroscopic protein dipole Langevin dipole method in its linear response approximation version (PDLD/S-LRA) while using the best fitted values of the dielectric constants ε′p and ε′eff for the self energy and charge–charge interactions, respectively. The method is validated on a diverse set of 45 proteins. It is found that the best fitted values of both dielectric constants are around 40. However, the self energy of internal residues and the charge–charge interactions of Lys have to be treated with care, using a somewhat lower values of ε′p and ε′eff. The predictions of ΔGfold reported here, have an average error of only 1.8 kcal/mole compared to the observed values, making our method very promising for estimating protein stability. It also provides valuable insight into the complex electrostatic phenomena taking place in folded proteins. Proteins 2009. © 2009 Wiley-Liss, Inc.