The conserved topological structure observed in various molecular families such as globins or cytochromes c allows structural equivalencing of residues in every homologous structure and defines in a coherent way a global alignment in each sequence family. A search was performed for equivalent residue pairs in various topological families that were buried in protein cores or exposed at the protein surface and that had mutated but maintained similar unmutated environments. Amino acid residues with atoms in contact with the mutated residue pairs defined the environment. Matrices of preferred amino acid exchanges were then constructed and preferred or avoided amino acid substitutions deduced. Given the conserved atomic neighborhoods, such natural in vivo substitutions are subject to similar constraints as point mutations performed in site-directed mutagenesis experiments. The exchange matrices should provide guidelines for “safe” amino acid substitutions least likely to disturb the protein structure, either locally or in its overall folding pathway, and most likely to allow probing of the structural and functional significance of the substituted site.