The effect of low concentrations of hydrogen peroxide (H2O2) (5 x 10(-7)-9.5 x 10(-7) M) on cell growth and antibody production was investigated with murine hybridoma cells (Mark 3 and anti-hPL) in culture. Cell growth, measured by flow cytometry with morphological parameters, was significantly stimulated by H2O2 (8 x 10(-7) M) but H2O2 concentration of 7 x 10(-6) M and above increased cell death. H2O2 stimulation of antibody production was nonsignificant. The metabolism of cells treated with 8 x 10(-7) or 1 x 10(-5) M H2O2 was similar to that of the control in terms of glucose and glutamine consumption, lactate and ammonia production, and amino acid concentrations in the medium. The concentrations of lactate dehydrogenase, a marker of cell death, in test and control cells were similar. However, concentrations of intracellular free radicals measured by flow cytometry with dihydrorhodamine 123 (DHR 123) and dichlorofluorescein diacetate (DCFH-DA) as fluorochromes were different. The reactive oxygen species content of cells in 8 x 10(-7) M H2O2 was similar to that of the controls, but there was a sudden, marked production of superoxide anions (detected with DHR 123) and H2O2 or peroxides (detected with DCFH-DA) by cells incubated with 1 x 10(-5) M H2O2 which increased with increasing H2O2 until cell death.