Background: GS-9148 is a novel structural analog of dAMP that acts as a nucleotide HIV reverse transcriptase (RT) inhibitor (NRTI). Both GS-9148 and its phosphonoamidate prodrug, GS-9131, exhibit potent activity in vitro against a range of NRTI-resistant HIV clinical isolates, including those with TAMs or K65R. In cell culture, GS-9148 selects for HIV mutations in RT at codon 70, where K70N develops first and is replaced by a triple mutant containing K70E/D123N/T165I. Methods: Susceptibilities of K70N, K70E, and K70E/D123N/T165I site-directed mutant viruses to NRTIs were determined in cell culture. The two known enzymatic mechanisms of resistance were assayed: NRTI binding or incorporation was measured by steady state kinetics (K_i/K_m) and ATP-mediated excision was measured with GS-9148- and other NRTI-terminated primers. Results: Viruses containing K70E plus D123N and/or T165I but not by K70N or K70E alone showed low-level decreased susceptibility to GS-9148 (3- to 4-fold) and other NRTIs (2- to 7-fold), but retained full susceptibility to AZT. Biochemically, the K70N, K70E, and K70E/D123N/T165I mutant RTs showed decreased binding or incorporation of all NRTIs examined (2.4-, 2.6-, and 4.0-fold increased K_i/K_m for GS-9148 diphosphate, respectively). GS-9148 exhibited low levels of excision by wild-type and K70E-containing RT, whereas K70E showed decreased excision of tenofovir and AZT-MP compared to wild-type. Conclusions: This mechanistic study confirmed the primary role of K70E in the resistance phenotype selected by GS-9148. For GS-9148 and other NRTIs, the resistance due to K70E was mediated primarily by decreased binding or incorporation of the inhibitors. The K70E mutants retained full susceptibility to AZT, where decreased incorporation of AZT-TP was counteracted by decreased AZT-MP excision. These data suggest that the K70E mutant utilizes a resistance mechanism similar to that of K65R, but shows lower levels of resistance against NRTIs.