Although the V118I mutation in reverse transcriptase causes a reduced incorporation of zidovudine and lamivudine into transcribed viral DNA, it also decreases ATP-mediated pyrophosphorylysis. In this manner, its overall impact on enzymatic activity appears to be neutral when assessed in vitro. Nevertheless, V118I remains identified as a resistance mutation by a number of commonly utilised resistance mutation algorithms. A large clinical database was queried to identify antiretroviral-naive patients with V118I as the sole resistance associated mutation. Each index patient was matched to five control patients with no major mutations who were prescribed an identical first-line regimen. The two groups were compared in terms of virological and immunological response up to 24 months after initiation of first-line antiretroviral therapy in an intent-to-treat analysis. The V118I mutation was detected as the sole major reverse transcriptase mutation in 35 (1.8%) samples. Twenty-five of these patients initiated antiretroviral therapy (between March 1997 and October 2002) and could therefore be included in the analysis of therapeutic response. Twenty of 25 patients were prescribed zidovudine and/or lamivudine. The index and control (n = 125) groups were well balanced with respect to demographic and clinical characteristics. There was no statistically significant difference in the average reduction in HIV RNA viral load (global P = 0.9) or in the average increase in CD4 cell count (global P = 0.5) compared to baseline. Our study suggests that V118I should be excluded from mutation lists used for clinical epidemiological studies of transmitted drug resistance. Furthermore, the presence of V118I as the sole nucleoside reverse transcriptase inhibitor mutation should not be over-interpreted when deciding on therapeutic options.