HIV-1 reverse transcriptase (RT) plays a critical role in the HIV lifecycle and is a major drug target. Although nonnucleoside reverse transcriptase inhibitors (NNRTIs) have proven effective anti-AIDS drugs, NNRTI resistance mutations appear frequently in patient populations and represent a continuing challenge in the treatment of AIDS. K103N is a common NNRTI resistance mutation that conveys high levels of resistance to multiple NNRTIs including the widely used drug efavirenz (EFV). Because NNRTIs are allosteric inhibitors, mechanisms of both inhibition and resistance may be mediated by conformational dynamics.Hydrogen/deuterium exchange reveals that the K103N mutation increases the structural flexibility of RT in portions of the polymerase domain of the p66 subunit, which contains the polymerase active site and the NNRTI binding pocket. We also find increased flexibility in parts of the RNAse H domain. Previous H/D exchange studies showed that EFV binding induced significant long-range suppression of molecular flexibility in wild-type RT, suggesting that this rigidification may contribute to NNRTI inhibition. In K103N, EFV binding has negligible effects on molecular flexibility except in the immediate vicinity of the binding site. As a result, K103N-EFV complex is more flexible than wild type-EFV complex throughout most of the structure, including regions thought to be essential for DNA translocation. EFV binding studies by equilibrium dialysis show that, while the binding affinity of the drug is reduced by a factor of 3 for K103N RT compared to wild type, K103N RT is saturated with EFV under the conditions of the H/D exchange experiments. We propose that the retention of essential molecular mobility even when bound to inhibitor contributes to NNRTI resistance in K103N. Our findings suggest that conformational flexibility may play a role in the resistance of other RT mutants to NNRTIs.