Effects of cell fixation procedures appropriate for flow cytometric analysis on the infectivity of human T lymphoblastoid H9 cells infected with human immunodeficiency virus-1 (HIV-1) were evaluated to provide guidelines for choosing cell treatments for potentially infectious samples. H9 cells experimentally infected with HIV-1 were treated by the test fixation procedure, washed, and cocultured with equal numbers of live, uninfected H9 cells. To estimate the reduction in infectivity due to the fixation procedure, dilution series of live infected H9 cells in uninfected H9 cells were simultaneously established in culture. Cell cultures were incubated 8-10 d, harvested, and evaluated for evidence of HIV-1 infection by the presence of cell-associated HIV-1 antigens and/or by the presence of particle-associated reverse transcriptase activity in cell culture supernatants. Thirty-minute fixation with formaldehyde (1.85%), methanol (absolute), methanol:acetone (1:1), or paraformaldehyde (0.5%) reduced the infectivity of HIV-1-infected H9 cells by greater than 99.99%. To the same degree, a multi-step fixation procedure utilizing formaldehyde and ethanol was effective in reducing HIV-1 infectivity. Conversely, the erythrocyte fixative dimethylsuberimidate at 3 micrograms/ml was ineffective in reducing HIV-1 infectivity.