Nucleos(t)ide reverse transcriptase inhibitors (NRTIs) form the backbone of most anti-HIV therapies. We have shown that 4' -ethynyl-2-fluoro-2' -deoxyadenosine (EFdA) is a highly effective NRTI; however, the reasons for the potent antiviral activity of EFdA are not well understood. Here we use a combination of structural, computational, and biochemical approaches to examine how substitutions in the sugar or adenine rings affect the incorporation of dA-based NRTIs such as EFdA into DNA by HIV RT and their susceptibility to deamination by adenosine deaminase (ADA). NMR spectroscopy studies of 4' -substituted NRTIs show that ethynyl or cyano groups stabilize the sugar ring in the C2' -exo/C3' -endo (North) conformation. Steady state kinetic analysis of the incorporation of 4' -substituted NRTIs by RT reveal a correlation between the North conformation of the NRTI sugar ring and efficiency of incorporation into the nascent DNA strand. Structural analysis and the kinetics of deamination by ADA demonstrate that 4' -ethynyl and cyano substitutions decrease the susceptibility of adenosine-based compounds to ADA through steric interactions at the active site. However, the major determinant for decreased susceptibility to ADA is the 2-halo substitution, which alters the pKa of N1 on the adenine base. These results provide insight into how NRTI structural attributes affect their antiviral activity through their interactions with the RT and ADA active sites.