We describe selective “fluorous” effects in the active site of a DNA polymerase, by using nucleotide analogues whose pairing edges are perfluorinated. The 5′-triphosphate deoxynucleotide derivatives of DNA base analogues 2,3,4,5-tetrafluorobenzene (FB) and 4,5,6,7-tetrafluoroindole (FI), as well as hydrocarbon controls benzene (B) and indole (I), were synthesized and studied as substrates for the DNA Polymerase I Klenow fragment (KF exo−). Modified nucleotides were present in the DNA template or were supplied as nucleoside triphosphates in studies of the steady-state kinetics of single nucleotide insertion. When supplied opposite the non-natural bases in the template strand, the hydrophobic nucleoside triphosphates were incorporated by up to two orders of magnitude more efficiently than the natural deoxynucleoside triphosphates. The purine-like fluorinated indole nucleotide (FI) was the most efficiently inserted of the four hydrophobic analogues, with the most effective incorporation occurring opposite the pyrimidine-like tetrafluorobenzene (FB). In all cases, the polyfluorinated base pairs were more efficiently processed than the analogous hydrocarbon pairs. A preliminary test of polymerase extension beyond these pairs showed that only the FB base is appreciably extended; the inefficient extension is consistent with recently published data regarding other nonpolar base pairs. These results suggest the importance of hydrophobicity, stacking, and steric interactions in the polymerase-mediated replication of DNA base pairs that lack hydrogen bonds. These findings further suggest that the enhanced hydrophobicity of polyfluoroaromatic bases could be employed in the design of new, selective base pairs that are orthogonal to the natural Watson–Crick pairs used in replication.