We describe a survey of protein-DNA interactions with seven different DNA polymerases and reverse transcriptases, carried out with nonpolar nucleoside isosteres F (a thymidine analog) and Z and Q (deoxyadenosine analogues). Previous results have shown that Z and F can be efficiently replicated opposite each other by the exonuclease-free Klenow fragment of DNA polymerase I from Escherichia coli (KF-), although both of them lack Watson-Crick II-bonding ability. We find that exonuclease-inactive T7 DNA polymerase (T7(-)), Thermus aquaticus DNA polymerase (Taq), and HIV-reverse transcriptase (HIV-RT) synthesize the nonnatural base pairs A-F, F-A, F-Z, and Z-F with high efficiency, similarly to KF-. Steady-state kinetics were also measured for T7(-) and the efficiency of insertion is very similar to that of KF-; interestingly, the replication selectivity with this pair is higher for T7- than KF-, possibly due to a tighter active site. A second group comprised of calf thymus DNA polymerase ct (Pol cc) and avian myeloblastosis virus reverse transcriptase (AMV-RT) was able to replicate the A-F and F-A base pairs to some extent but not the F-Z and the Z-F base pairs. Most of the insertion was recovered when Z was replaced by the nucleoside Q (9-methyl-1-H-imidazo[(4,5)-b]pyridine), which is analogous to Z but possesses a minor groove acceptor nitrogen. This strongly supports the existence of an energetically important hydrogen-bonded interaction between the polymerase and the minor groove at the incipient base pair for these enzymes. A third group, formed by human DNA polymerase beta (Pol beta) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT), failed to replicate the F-Z and Z-F base pairs. No insertion recovery was observed when Z was replaced by Q, possibly indicating that hydrogen bonds are needed at both the template and the triphosphate sites. The results point out the importance of DNA minor groove interactions at the incipient base pair for the activity of some polymerases, and demonstrate the variation in these interactions from enzyme to enzyme.