Antiretroviral (ARV) treatment decisions are difficult for HIV-1-infected patients on complex treatment regimens who have partial suppression of HIV-1 replication and limited treatment options. Information on the ARV activity of the components of a complex regimen would be useful. Sixteen subjects who had received prolonged therapy with zidovudine (ZDV) and lamivudine (3TC), with a median duration of 32.5 months, were discontinuing this dual-nucleoside regimen and volunteered to have plasma HIV-1 RNA levels monitored over the 2 weeks after discontinuation. All subjects experienced an increase in HIV-1 RNA after discontinuation, with a median increase of 0.54 log10 copies/mL over 2 weeks (range: 0.31-1.71; P < 0.001). An inverse correlation existed between the decline in HIV-1 RNA levels over 2 to 3 years on nucleoside analogue therapy and the increase over the 10 to 14 days off therapy (Spearman r = -0.53; P = 0.036). Over the 2-week period, a subset of individuals who had genotype testing at multiple reverse transcriptase codons associated with ZDV and 3TC resistance had no changes in genotype off therapy. Nucleoside analogue reverse transcriptase inhibitors may have continued ARV activity despite long durations of partially suppressive therapy and the presence of resistant HIV-1.