The HIV-1 reverse transcriptase (RT) resistance mutations K65R and M184V occur individually and in combination, and can contribute to decreased treatment responses in patients. In order to understand how these mutations interact with one another to confer drug resistance, the susceptibilities and underlying resistance mechanisms of these mutants to nucleoside RT inhibitors (NRTIs) were determined. Virus carrying K65R have reduced susceptibility to most NRTIs, but retain full susceptibility to zidovudine (AZT). M184V mutants have reduced susceptibility to lamivudine (3TC), emtricitabine (FTC) and didanosine (ddl), and contribute to reduced susceptibility to abacavir; however, they remain fully susceptible to tenofovir (TFV), AZT and stavudine (d4T). In cell culture, the K65R+M184V virus showed slightly increased susceptibility to TFV, AZT and d4T compared with K65R alone, but showed further decreases in susceptibility to 3TC, FTC, ddl and abacavir. There are two major biochemical mechanisms of resistance: altered NRTI binding/incorporation and altered NRTI excision after incorporation. For most NRTIs, the primary mechanism of resistance by K65R, M184V and K65R+M184V mutant RTs is to disrupt the NRTI-binding/incorporation steps. In the case of AZT, however, decreased binding/incorporation by K65R and K65R+M184V was counteracted by decreased AZT excision resulting in wild-type susceptibility. For TFV, decreased excision by K65R and K65R+M184V may partially counteract the K65R-driven decrease in incorporation relative to wild-type resulting in only low levels of TFV resistance. The K65R-mediated effect on decreasing NRTI excision was stronger than for M184V. These studies show that both mechanisms of resistance (binding/incorporation and excision) must be considered when defining resistance mechanisms.