Efavirenz is a potent and selective nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Nucleotide sequence analyses of the protease and RT genes (coding region for amino acids 1 to 229) of multiple cloned HIV-1 genomes from virus found in the plasma of patients in phase II clinical studies of efavirenz combination therapy were undertaken in order to identify the spectrum of mutations in plasma-borne HIV-1 associated with virological treatment failure. A K103N substitution was the HIV-1 RT gene mutation most frequently observed among plasma samples from patients for whom combination therapy including efavirenz failed, occurring in at least 90% of cases of efavirenz-indinavir or efavirenzzidovudine (ZDV)-lamivudine (3TC) treatment failure. V108I and P225H mutations were observed frequently, predominantly in viral genomes that also contained other nonnucleoside RT inhibitor (NNRTI) resistance mutations. L100I, K101E, K101Q, Y188H, Y188L, G190S, G190A, and G190E mutations were also observed. V106A, Y181C, and Y188C mutations, which have been associated with high levels of resistance to other NNRTIs, were rare in the patient samples in this study, both before and after exposure to efavirenz. The spectrum of mutations observed in cases of virological treatment failure was similar for patients initially dosed with efavirenz at 200, 400, or 600 mg once a day and for patients treated with efavirenz in combination with indinavir, stavudine, or ZDV-3TC. The proportion of patients carrying NNRTI resistance mutations, usually K103N, increased dramatically at the time of initial viral load rebound in cases of treatment failure after exposure to efavirenz. Viruses with multiple, linked NNRTI mutations, especially K103N-V108I and K103N-P225H double mutants, accumulated more slowly following the emergence of K103N mutant viruses.The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is critical to the life cycle of HIV and is without a homologue in eukaryotic organisms. As such, it is an attractive target for selective antiviral therapy. Among inhibitors of RT, a large class of chemically diverse, generally HIV-1-specific, nonnucleoside reverse transcriptase inhibitors (NNRTIs) has been identified. These inhibitors generally act by binding to a site on the RT that is distinct from the polymerase catalytic site. While NNRTIs can be potent inhibitors of HIV-1 replication, with favorable safety and pharmacokinetic parameters, rapid emergence of resistant viruses both in vitro (20, 24) and in vivo (11,14,15,25,30), often as the result of single nucleotide changes, has limited the therapeutic utility of these compounds as monotherapy. However, recent clinical trials of the use of NNRTIs in combination with other antiretroviral agents have demonstrated an added benefit from inclusion of