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Markers of maturation and activation were measured on peripheral CD4+ T cells in chronically HIV-1–infected patients in a randomized, controlled pilot study of structured treatment interruption (STI). Eight subjects underwent 2 cycles of 1 month off and 1 month on highly active antiretroviral therapy (HAART), followed by a final 3-month interruption. During STI, CD4+ T-cell percentage remained relatively stable in 4 of 8 subjects. The remaining 4 STI subjects had significant rapid decline in CD4+ T-cell percentage during STI, followed by return to pre-STI baseline while on HAART. Changes in overall CD4+ T-cell percentage corresponded with fluctuations in the CD45RA+CCR7+ naive and CD45RA−CCR7+ central memory subsets. Subjects with variable CD4+ T-cell percentages tended to have higher pre-HAART plasma HIV-1 RNA setpoints and experienced higher levels of plasma HIV-1 RNA rebound during STI. These results suggest that interruptions should be avoided whenever possible in patients on HAART with high plasma HIV-1 RNA setpoints.