AbstractThe concept of hydrophobicity is critical to our understanding of the principles of membrane protein (MP) folding, structure, and function. In the last decades, several groups have derived hydrophobicity scales using both experimental and statistical methods that are optimized to mimic certain natural phenomena as closely as possible. The present work adds to this toolset the first knowledge‐based scale that unifies the characteristics of both α‐helical and β‐barrel multispan MPs. This unified hydrophobicity scale (UHS) distinguishes between amino acid preference for solution, transition, and trans‐membrane states. The scale represents average hydrophobicity values of amino acids in folded proteins, irrespective of their secondary structure type. We furthermore present the first knowledge‐based hydrophobicity scale for mammalian α‐helical MPs (mammalian hydrophobicity scale—MHS). Both scales are particularly useful for computational protein structure elucidation, for example as input for machine learning techniques, such as secondary structure or trans‐membrane span prediction, or as reference energies for protein structure prediction or protein design. The knowledge‐based UHS shows a striking similarity to a recent experimental hydrophobicity scale introduced by Hessa and coworkers (Hessa T et al., Nature 2007;450:U1026–U1032). Convergence of two very different approaches onto similar hydrophobicity values consolidates the major differences between experimental and knowledge‐based scales observed in earlier studies. Moreover, the UHS scale represents an accurate absolute free energy measure for folded, multispan MPs—a feature that is absent from many existing scales. The utility of the UHS was demonstrated by analyzing a series of diverse MPs. It is further shown that the UHS outperforms nine established hydrophobicity scales in predicting trans‐membrane spans along the protein sequence. The accuracy of the present hydrophobicity scale profits from the doubling of the number of integral MPs in the PDB over the past four years. The UHS paves the way for an increased accuracy in the prediction of trans‐membrane spans. Proteins 2009. © 2008 Wiley‐Liss, Inc.