The kinetics of forming all possible single base substitution errors are measured for Drosophila melanogaster DNA polymerase alpha and avian myeloblastosis virus reverse transcriptase. Seventeen sites along bacteriophage M13 DNA are investigated so that effects of nearest neighbor base stacking on misinsertion kinetics can be evaluated. Polymerase alpha appears to be more error prone than reverse transcriptase. Polymerase alpha forms transversion mispairs at rates comparable to transition mispairs with two exceptions; A.A and C.C are formed with significantly higher and lower efficiencies, respectively. Reverse transcriptase forms transversions with lower efficiencies than transitions, especially low being A.G, G.G, and C.C. For both enzymes, misinsertion frequencies vary typically by 10-fold for the same mispair in different locations. Misinsertion frequency can be expressed as a product of two components, one based on Km and the other on Vmax. DNA polymerase alpha appears to use primarily Km discrimination (100-5000-fold) to achieve insertion fidelity while reverse transcriptase shows a greater balance between Km and Vmax discrimination. Nearest-neighbor base stacking interactions appear to have opposite effects on the two discrimination components. The 5'-nearest neighbor influence on Km is greater for correct insertions than for incorrect, while the influence on Vmax is greater for the incorrect base. Target sites that have pyrimidine as the 5'-nearest neighbor to incoming nucleotides show a higher than average misinsertion component based on Km, but a lower than average component based on Vmax. Conversely, target sites with nearest neighbor purines have a higher than average Vmax component. These results imply that nucleotide misinsertion "hot spots" will occur next to pyrimidines when Km discrimination is dominant and next to purines when Vmax discrimination is dominant. When Vmax and Km discrimination components have similar magnitudes, nearest neighbor effects tend to cancel thereby reducing the effects of base stacking on insertion error rates.