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To analyse the presence of genotypic and phenotypic resistance in lymph node mononuclear cells from patients with sustained undetectable plasma HIV-1 RNA with highly active antiretroviral therapy.Cross-sectional study on 27 HIV-infected patients receiving triple therapy for a mean period of 232.8 ± 22.1 weeks.HIV-1 RNA was measured in plasma and lymph node cells using PCR. Reverse transcriptase and protease genes were sequenced from HIV-1 RNA obtained from lymph node cells and from peripheral blood mononuclear cell proviral DNA using a commercially available kit (TruGene). Phenotypic resistance was assessed by using a recombinant virus assay (AntiVirogram).Mutations were not found in lymph node mononuclear cell RNA in six out of nine patients on first-line regimens although they were detected in 15 out of 18 who received prior suboptimal combinations. Phenotypic resistance was confirmed in most of these cases. These patterns of resistance were closely related to patients’ history of antiretroviral therapy and genotypic analysis of plasma HIV-1 RNA taken just before initiation of the current regimen. In half the patients analysed, resistance mutations found in lymph nodes were not always detected in archival proviral DNA from blood cells. Mean levels of HIV-1 RNA in lymph node cells were not different in patients exhibiting resistance compared with those harbouring wild-type viruses.These data demonstrate that resistant HIV-1 is produced in lymphoid tissues for prolonged periods despite effective therapy. The mechanism could represent a release from previously infected cells rather than new cycles of cellular infection.