OBJECTIVE To investigate the effects of salvage therapy with lopinavir-ritonavir on HIV-1 phenotype in heavily antiretroviral experienced HIV-infected children. METHODS Twenty antiretroviral experienced HIV-infected children were studied during a mean of time of 16.1 months from initiation of the treatment with lopinavir-ritonavir. METHODS Besides CD4 T cells, viral load and clinical status, we analyzed 91 serial viral isolates to study the phenotype, and biological clones derived from co-cultivation techniques. RESULTS We observed an increase in CD4 T cells, a statistically significant decrease in viral load and clinical benefits from 3 months after treatment. Ninety per cent of children had SI/X4 bulk isolates in peripheral blood mononuclear cells at study entry. The viral phenotype changed to non syncitium-inducing (NSI)/R5 in 94% of the children after a mean of 5.7 months (95% confidence interval, 2.1-9.3 months) of salvage therapy. The remaining 10% of children had NSI/R5 isolates at entry and at all follow-up study. Similar results were found at the clonal level. Thus, at study entry in PBMC of three children with bulk syncitium-inducing (SI) phenotype, we recovered 65 biologic clones, 56 being SI and nine NSI. After salvage therapy bulk isolates changed to NSI and of 40 biologic clones recovered only five were SI and the rest were NSI. CONCLUSIONS Our data suggest that lopinavir-ritonavir salvage therapy led not only to a viral load decrease but also to a phenotypic change. X4 virus appeared to be preferentially suppressed. Shifts in co-receptor usage may thereby contribute to the clinical efficacy of anti-HIV drugs in vertically infected infants.