We previously identified a rare mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), I132M, which confers high-level resistance to the nonnucleoside RT inhibitors (NNRTI) nevirapine and delavirdine. In this study, we have further characterized the role of this mutation in viral replication capacity and in resistance to other RT inhibitors. Surprisingly, our data show that I132M confers marked hypersusceptibility to the nucleoside analogs lamivudine (3TC) and tenofovir, both at the virus and enzyme level. Subunit-selective mutagenesis studies revealed that the mutation in the p51 subunit of RT was responsible for increased sensitivity to the drugs, and transient kinetic analyses showed that this hypersusceptibility was due to I132M decreasing the enzyme s affinity for the natural dCTP substrate but increasing its affinity for 3TC-triphosphate. Furthermore, the replication capacity of HIV-1 containing I132M is severely impaired. This decrease in viral replication capacity could be partially or completely compensated by the A62V or L214I mutation, respectively. Taken together, these results help to explain the infrequent selection of I132M in patients failing NNRTI regimens, and furthermore demonstrate that a single mutation outside of the polymerase active site, and in the p51 subunit of RT, can significantly influence nucleotide selectivity.