The influence of the stability of base pairs formed by nearest-neighbor nucleotides on misincorporation frequency has been studied with the large fragment of DNA polymerase I, the alternating DNA copolymers, poly(dI-dC) and poly-(dG-dC), as template-primers, and dGTP, dITP, and dCTP as substrates. We have utilized the difference in thermodynamic stability between the doubly H-bonded I X C base pair and triply H-bonded G X C base pair to examine the effects of base-pair stability of both the "preceding" and the "following" nucleotides on the frequency of insertion of a mismatched nucleotide, as well as on its stable incorporation into polynucleotide. The present studies demonstrate that the stability of the base pairs formed by nearest-neighbor nucleotides affects the frequency of incorporation of noncomplementary nucleotides. Misincorporation frequency is increased when the nearest-neighbor nucleotides form more stable base pairs with the corresponding nucleotides in the template and is decreased when they form less stable base pairs. The stability of the base pair formed by a nucleotide either preceding (5' to) or following (3' to) a misincorporated nucleotide influences misincorporation frequency, but by different mechanisms. The stability of base pairs formed by preceding nucleotides affects the rate of insertion of mismatched nucleotide but does not protect the mismatched nucleotide from removal by the 3' to 5' exonuclease activity. In contrast, the stability of a base pair formed by a following nucleotide determines whether a misincorporated nucleotide is extended or excised by affecting the ability of the enzyme to edit errors of incorporation.