Human immunodeficiency virus (HIV) type 1 DNA in peripheral blood mononuclear cells (PBMC) was quantified in 31 children who received efavirenz, nelfinavir, and 1 or 2 nucleoside reverse-transcriptase inhibitors for > or =2 years and in whom undetectable plasma HIV-1 RNA levels (< 50 copies/mL) were sustained, to determine the usefulness of HIV-1 DNA as a marker of virus suppression. The median baseline HIV-1 DNA level was 750 copies/10(6) PBMC. After initiation of highly active antiretroviral therapy (HAART), HIV-1 DNA levels decreased gradually, reaching a plateau from week 80 through week 104 (median HIV-1 DNA level, 263 copies/10(6) PBMC). Children who had plasma HIV-1 RNA levels < 50 copies/mL after receiving HAART for 8 weeks (n=16) had persistently lower quantities of intracellular HIV-1 DNA than children whose HIV-1 RNA levels reached < 50 copies/mL after 8 weeks of HAART (n=15). The median half-life for intracellular HIV-1 DNA was 60 weeks. Thus, despite prolonged maintenance of undetectable levels of plasma HIV-1 RNA, HIV-1 DNA remains detectable in PBMC of children and may be a useful marker of further virus suppression.