The long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) contains sequences required for the initiation of gene transcription. Among the substances known to activate the HIV-1 LTR is hydrogen peroxide (H202). We report here that H₂O₂-induced activation of the LTR in the macrophage cell line THP-1 and the lymphocyte cell line, Jurkat, is greatly increased by vanadate. Activation of the LTR by phorbol myristate acetate, tumor necrosis factor alpha, lipopolysaccharide, or Staphylococcus epidermidis extract was not increased by vanadate, indicating some selectivity for H₂O₂. H₂O₂ and vanadate also acted synergistically to increase the production of HIV-1 virions by the latently infected macrophage cell line U-1 as determined by p24 antigen release and the detection of intact virions by electron microscopy. Effects were observed at H₂O₂ and vanadate concentrations down to 3 × 10⁻⁶, with high concentrations leading to cell toxicity. Catalase was strongly inhibitory when added prior to the interaction of H₂O₂ and vanadate, but was considerably less inhibitory when the H₂O₂ and vanadate were allowed to preincubate prior to the catalase addition. H₂O₂ reacts with vanadate to form peroxides of vanadate that have potent biological effects. Our findings suggest that among these is the activation of the HIV-1 LTR.