Summary This study determines levels of regulatory T cells (T(regs)), naive T(regs), immune activation and cytokine patterns in 15 adult human immunodeficiency virus (HIV)-infected patients receiving prolonged highly active anti-retroviral therapy (HAART) who have known thymic output, and explores if naive T(regs) may represent recent thymic emigrant T(regs). HIV-infected patients treated with HAART with a median of 1 and 5 years were compared with healthy controls. Percentages of T(regs) (CD3(+)CD4(+)CD25(+)CD127(low)), naive T(regs) (CD3(+)CD4(+)CD25(+)CD45RA(+)) and activation markers (CD38(+)human leucocyte antigen D-related) were determined by flow cytometry. Forkhead box P3 mRNA expression and T cell receptor excision circles (T(REC)) content in CD4(+) cells were determined by polymerase chain reaction and cytokines analysed with Luminex technology. Levels of T(regs) were significantly higher in HIV-infected patients compared with controls, both after 1 and 5 years of HAART (P < 0.001), despite fully suppressed HIV-RNA and normalization of both CD4 counts, immune activation and cytokine patterns. Furthermore, levels of naive T(regs) were elevated significantly in HIV-infected patients (P < 0.001) and were associated with thymic output measured as the T(REC) frequency in CD4(+) cells (P = 0.038). In summary, T(reg) levels in HIV-infected patients are elevated even after 5 years of HAART. Increased thymic production of naive T(regs) may contribute to higher T(reg) levels in HIV-infection.