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Reverse transcriptases (RTs) are typically assayed in vitro with 5-10 mM Mg 2+ , whereas the free Mg 2+ concentration in cells is much lower. Artificially high Mg 2+ concentrations used in vitro can misrepresent different properties of human immunodeficiency virus (HIV) RT, including fidelity, catalysis, pausing, and RNase H activity. Here, we analyzed nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in primer extension assays at different concentrations of free Mg 2+ . At low concentrations of Mg 2+ , NRTIs and dideoxynucleotides (AZTTP, ddCTP, ddGTP, and 3TCTP) inhibited HIV-1 and HIV-2 RT synthesis less efficiently than they did with large amounts of Mg 2+ , whereas inhibition by the "translocation-defective RT inhibitor" EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) was unaffected by Mg 2+ concentrations. Steady-state kinetic analyses revealed that the reduced level of inhibition at low Mg 2+ concentrations resulted from a 3-9-fold (depending on the particular nucleotide and inhibitor) less efficient incorporation (based on k cat /K m ) of these NRTIs under this condition compared to incorporation of natural dNTPs. In contrast, EFdATP was incorporated with an efficiency similar to that of its analogue dATP at low Mg 2+ concentrations. Unlike NRTIs, NNRTIs (nevirapine, efavirenz, and rilviripine), were approximately 4-fold (based on IC 50 values) more effective at low than at high Mg 2+ concentrations. Drug-resistant HIV-1 RT mutants also displayed the Mg 2+ -dependent difference in susceptibility to NRTIs and NNRTIs. In summary, analyzing the efficiency of inhibitors under more physiologically relevant low-Mg 2+ conditions yielded results dramatically different from those from measurements using commonly employed high-Mg 2+ in vitro conditions. These results also emphasize differences in Mg 2+ sensitivity between the translocation inhibitor EFdATP and other NRTIs.