Human T-cell lines and normal lymphocytes persistently or acutely co-infected with the human immunodeficiency virus type 1 (HIV-1) and mycoplasmas were found to release hydrogen peroxide (H2O2), a likely cause of oxidative stress in these cells. The spectrofluorometric measurement of H2O2 release from these cells, using the scopoletin fluorescence quenching technique, gave values of 16-84 p moles/10(6) cells/min. In CEM cells, H2O2 was released only when acutely co-infected with HIV-1 and mycoplasmas, and not when infected with either organism alone. Anti-mycoplasmal antibiotics strongly reduced H2O2 release, and improved cell viability without blocking virus replication. These results suggest that the simultaneous infection by HIV-1 and mycoplasma leads to the release of H2O2, a toxic and potentially lethal metabolite, which in vivo may contribute to HIV-1 pathogenicity.