Background: Enfuvirtide, a peptide inhibitor of human immunodeficiency virus (HIV)-1-host cell membrane fusion, is the first of a new class of antiretroviral agents, the entry inhibitors. The safety and antiretroviral activity of enfuvirtide treatment of 24 weeks in HIV-1-infected children has been previously documented. Here we present the long term tolerability and safety of enfuvirtide. Methods: Fourteen children, 4 to 12 years of age, with incompletely suppressed HIV-1 infection were evaluated. Enfuvirtide was administered twice daily by subcutaneous injection. After the first 24 weeks of enfuvirtide dosing, subjects were evaluated every 8 weeks up to 96 weeks of therapy. At each visit, each subject had a physical examination and an assessment for adverse events with particular attention to evaluation of injection site reactions. Laboratory studies obtained at each visit included hematology and blood chemistry values, plasma HIV-1 RNA concentrations and CD4+ T cell counts. Results: Of 14 subjects, 6 completed at least 96 weeks of treatment. One child discontinued enfuvirtide after 22 days of treatment because of an aversion to injections, and 1 child electively discontinued after week 24 because of surgical complications unrelated to study drug. Four subjects discontinued study because of virologic failure, defined as an increase or persistence of plasma HIV-1 RNA ≥ 1.0 log10 copies/mL above baseline, which occurred between weeks 40 and 63. Two children experienced grade 3 adverse events resulting in discontinuation of the study drug; 1 subject developed grade 3 thrombocytopenia and 1 developed grade 3 edema at weeks 65 and 77, respectively. Eleven of 14 children had local injection site reactions during the first 24 weeks of treatment, 4 of the 12 subjects who continued treatment beyond week 24 reported local reactions. Generally, these local reactions were 1- to 3-cm tender nodules that developed after the injections and lasted for 1-2 days. Twelve children developed new diagnoses during treatment with enfuvirtide. None was judged to be definitively related to the study drug. Thirty-six percent of children starting enfuvirtide had HIV-1 RNA levels >1 log10 copies/mL below baseline levels at week 96. Children remaining on enfuvirtide for the entire 96 weeks had a median of 65 cells/mm3 and 9% increase in CD4+ T cells. Conclusions: Enfuvirtide was generally safe and, except for a high rate of injection site reactions, well-tolerated in HIV-1-infected children for as long as 96 weeks.