The emergence of drug resistance-conferring mutations can severely compromise the success of chemotherapy directed against human immunodeficiency virus type 1 (HIV-1). The M184V and/or L74V mutation in the reverse transcriptase (RT) gene are frequently found in viral isolates from patients treated with the nucleoside RT inhibitors lamivudine (3TC), abacavir (ABC), and didanosine (ddI). However, the effectiveness of combination therapy with regimens containing these compounds is often not abolished in the presence of these mutations; it has been conjectured that diminished fitness of HIV-1 variants containing L74V and M184V may contribute to sustained antiviral effects in such cases. We have determined that viruses containing both L74V and M184V are more impaired in replication capacity than viruses containing either mutation alone. To understand the biochemical mechanisms responsible for this diminished fitness, we generated a series of recombinant mutated enzymes containing either or both of the L74V and M184V substitutions. These enzymes were tested for their abilities to bypass important rate-limiting steps during the complex process of reverse transcription. We studied both the initiation of minus-strand DNA synthesis with the cognate replication primer human tRNA(3)(Lys) and the initiation of plus-strand DNA synthesis, using a short RNA primer derived from the viral polypurine tract. We observed that the efficiencies of both reactions were diminished with enzymes containing either L74V or M184V and that these effects were significantly amplified with the double mutant. We also show that release from intrinsic pausing sites during reverse transcription appears to be a major obstacle that cannot be efficiently bypassed. Our data suggest that the efficiency of RNA-primed DNA synthesis represents an important consideration that can affect viral replication kinetics.