The Leu-74 to Val (Leu74Val) mutation in human immunodeficiency virus type 1 reverse transcriptase (RT) develops as a consequence of didanosine (ddI) therapy and is associated with a decreased susceptibility to ddI. In this report, we provide evidence that the ddI-associated Leu74Val mutation confers a replication disadvantage to the virus. In a series of experiments, we have shown that (i) a cloned virus with an engineered Leu74Val mutation in RT was attenuated for replication; (ii) a Val-to-Leu revertant of Leu74Val in the pNL4-3 background replicated with an efficiency similar to that of the wild-type virus; (iii) when two isolates from the same patient were compared, a clinical isolate containing mutations Leu74Val and Thr215Tyr was attenuated for replication compared to one in which the Thr215Tyr mutation alone was present; and (iv) the viruses with the Leu74Val mutation showed an 11% loss of fitness in a single passage compared to the wild-type and a mutant virus containing a Lys70Arg mutation. The loss of fitness for viruses grown in drug-free medium could result in an inability to detect a Leu74Val mutant in clinical isolates obtained post-ddI therapy. The decreased replication ability of the Leu74Val mutant virus selected by ddI therapy provides a strong rationale for the lower viral RNA levels observed with ddI therapy compared to zidovudine therapy in clinical trials.