The incorporation of the deoxyribonucleotide of 2-aminopurine [(AP)] for deoxyadenylate into deoxyribonucleic acid (DNA) in vitro has been examined by using five highly purified DNA polymerases: calf thymus polymerase alpha, Escherichia coli polymerase I, and the polymerases induced by T4 phage mutant L56 (mutator phenotype), wild-type T4 phage, and T4 phage mutant L141 (antimutator phenotype). On a template of gapped salmon sperm DNA, the overall incorporation of (AP) relative to the incorporation of adenine decreases in this series of enzymes, in line with the increasing 3'-exonucleolytic activity associated with these polymerases. The nearest-neighbor distributions for (AP) and for adenine in the newly synthesized DNA were determined to test for potential sequence selectivity in the incorporation of (AP). In polymerizations in which d(AP)TP fully replaced dATP, the L141 polymerase, and to a lesser degree the wild type T4 polymerase, synthesized a DNA in which the distribution for (AP) was distinctly skewed compared to the nearest-neighbor distribution observed for adenine; incorporation of (AP) was relatively favored after guanine and disfavored after adenine and thymine. These sequence effects were less pronounced in syntheses in which both dATP and d(AP)TP were present. When dGTP was replaced by dITP, or dTTP by dUTP, adenine was still incorporated to the normal extent after the analogue, but the incorporation of (AP) was reduced after these analogues, which form weaker base pairs. The results indicate that incorporation of (AP) is disfavored with all polymerases tested and that a greater bias exists with those polymerases containing an active 3'-exonuclease. This bias against (AP) incorporation is alleviated after strong base pairs, and particularly following guanine, possibly due to stabilizing vertical stacking interactions.