The effects of nearest neighbor interactions between a nucleotide base at the primer 3'-terminus and an incoming deoxyribonucleoside triphosphate on DNA polymerase catalyzed insertion were examined. Kinetics of inserting the fluorescent nucleotide analog 2-aminopurine deoxyribonucleotide (dAPMP) and dAMP opposite a template T by 3'-->5' exonuclease-deficient mutants of Klenow fragment (KF-) were measured on primer/templates of identical sequence except for the base pair at the 3'-primer terminus. In addition to its fluorescence properties, 2-aminopurine (AP) is an attractive probe because it is misinserted opposite T by polymerases at much higher frequencies than natural nucleotides. Misinsertion frequencies for AP are on the same order of magnitude as variations in misinsertion frequencies due to changes in local DNA sequence, which makes the statistical significance of these variations easier to document. We have established that changes in the fluorescence of AP can be used to follow the insertion of dAPMP on both steady-state and pre-steady-state time scales. Rates of insertion of dAPMP measured by fluorescence and by a polyacrylamide gel assay were similar and are sensitive to the identity of the base at the 3'-primer twice as fast as insertion following a primer terminus T. The difference in rates arises primarily from differences in kcat values, which were fastest next to G and slowest next to T, while apparent Km values were similar next to each of the 4 different nearest neighbors. The gel assay was used to measure AP misinsertion efficiencies by two methods: (1) by having dAPTP and dATP directly compete for insertion opposite T in the same reaction and (2) by measuring Vmax/Km values for each substrate in separate reactions. The results from the direct competition and separate kinetics measurements are similar. The misinsertion efficiency of dAPMP relative to dAMP opposite a template T was significantly higher next to a 3'-primer terminus G (f(ins) = 0.31 +/- 0.06) than next to T (f(ins) = 0.15 +/- 0.03) for the KF- single mutant (D42A). The corresponding misinsertion efficiencies next to a 3'-primer terminus G and T were 0.20 +/- 0.02 and 0.16, respectively, for the KF- double mutant (D355A, E357A). Relative rates of insertion of dAPMP and dAMP correlate with melting temperatures calculated for nearest neighbor doublets which reflect the relative base-stacking energies. In addition to changes in insertion kinetics, polymerase-DNA dissociation rates varied with the identity of the 3'-primer terminus, differing by as much as 7-20-fold depending on the polymerase and the primer/template.