The codon substitution matrix presented here is to our knowledge the first based entirely on empirical **data** and can serve in many fields of computational biology. We have found that at long distances, when the synonymous mutations have reached saturation, amino acid matrices are better suited for alignments and long-distance homology searching. Codon mutation matrices have advantages for alignment up to medium evolutionary distances and for usages that require DNA such as ancestral reconstruction of DNA sequences and the calculation of Ka/Ks ratios.