To better understand the energetics of accurate DNA replication, we directly measured Delta G degrees for the incorporation of a nucleotide into elongating dsDNA in solution (Delta G degrees(incorporation)). Direct measurements of the energetic difference between synthesis of correct and incorrect base pairs found it to be much larger than previously believed (average Delta Delta G degrees(incorporation) = 5.2 +/- 1.34 kcal mol(-1)). Importantly, these direct measurements indicate that Delta Delta G degrees(incorporation) alone can account for the energy required for highly accurate DNA replication. Evolutionarily, these results indicate that the earliest polymerases did not have to evolve sophisticated mechanisms to replicate nucleic acids; they may only have had to take advantage of the inherently more favorable Delta G degrees for polymerization of correct nucleotides. These results also provide a basis for understanding how polymerases replicate DNA (or RNA) with high fidelity.