Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent and commonly prescribed antiviral agents used in combination therapy (CART) of human immunodeficiency virus type 1 (HIV-1) infection. The development of drug resistance is a major limitation of CART. Reverse transcriptase (RT) genotypes with the NNRTI resistance mutations K101E؉G190S are highly resistant to efavirenz (EFV) and can develop during failure of EFV-containing regimens in patients. We have previously shown that virus with K101E؉G190S mutations can replicate more efficiently in the presence of EFV than in its absence. In this study, we evaluated the underlying mechanism for drug-dependent stimulation, using a single-cycle cell culture assay in which EFV was added either during the infection or the virus production step. We determined that EFV stimulates K101E؉G190S virus during early infection and does not affect late steps of virus replication, such as increasing the amount of active RT incorporated into virions. Additionally, we showed that another NNRTI, nevirapine (NVP), stimulated K101E؉G190S virus replication during the early steps of infection similar to EFV, but that the newest NNRTI, etravirine (ETR), did not. We also showed that EFV stimulates K101E؉Y188L and K101E؉V106I virus, but not K101E؉L100I, K101E؉K103N, K101E؉Y181C, or K101E؉G190A virus, suggesting that the stimulation is mutation specific. Real-time PCR of reverse transcription intermediates showed that although the drug did not stimulate minus-strand transfer, it did stimulate minus-strand strong-stop DNA synthesis. Our results indicate that stimulation most likely occurs through a mechanism whereby NNRTIs stimulate priming or elongation of the tRNA.The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) converts viral genomic RNA into doublestranded DNA through the process of reverse transcription (reviewed in reference 42). The enzyme is crucial for viral replication and has been an important target of antiretroviral therapy since 1987 (15). Efavirenz (EFV) is a nonnucleoside RT inhibitor (NNRTI) that is commonly used in combination with two nucleoside analog RT inhibitors (NRTIs) for the treatment of antiretroviral-agent-naïve patients (36a). NNRTIs inhibit virus replication by preventing DNA polymerization by RT, but EFV has a low barrier to resistance, because a single mutation can cause high-level drug resistance (1, 2). An interesting observation first published by our research group was that viral variants having the NNRTI resistance mutation combination K101EϩG190S replicated more efficiently in the presence of EFV than in its absence, suggesting that some variants selected by drug treatment were not only resistant but were actually stimulated in the presence of drug (39). The stimulation observed for the double mutant, K101EϩG190S, was seen despite the fact that the single K101E and G190S mutants did not show any stimulation. The K101EϩG190S double mutant is seen in 3 to 4% of patients failing EFV and is highly resistant to nevirapine ...