We studied the phagocytes from three infants with malignant osteopetrosis and from their families in an attempt to define further the phagocyte abnormalities associated with this disorder. The rapid membrane potential depolarization in response to the soluble stimuli formylmethionylleucylphenylalanine (FMLP) and phorbol myristate acetate (PMA) served as a measure of neutrophil activation, with 3,3-dipentyloxacarbocyanine (diOC5 [3]) used as the probe. A fluorescence-activated cell sorter (FACS) allowed us to use small volumes of blood in a new quantitative evaluation of neutrophil response. The neutrophils from the infants with malignant osteopetrosis were very minimally activated with either stimulus, as demonstrated by incomplete membrane potential depolarization (10% to 15% of normal controls). This finding indicates that malignant osteopetrosis is accompanied by a significantly reduced neutrophil response to stimulation. The abnormal activation process was also reflected in the respiratory burst response of the patients' neutrophils and monocytes. Fifty percent to 60% of the infants' neutrophils totally failed to reduce nitro blue tetrazolium dye (NBT), 30% to 40% of the cells showed only slight reduction after PMA or FMLP stimulation, and only 5% to 10% demonstrated normal reduction. Peripheral blood monocytes failed to reduce NBT in 35% to 70% of the cells tested. Similar testing of granulocyte-macrophage colonies grown in vitro from circulating progenitor cells also showed an abnormal distribution of response to PMA, with a majority of colonies showing a decrease or absence of NBT reduction. Thus control of expression of the osteopetrotic defect occurs at or before the progenitor cell level.