Cytochalasin B can influence various functions of human polymorphonuclear leukocytes, including chemotaxis, lysosomal enzyme release, and reactive oxygen species generation. In this study we investigated the effect of cytochalasin B on the increase in intracellular free calcium concentration after three consecutive additions of 10(-7) M N-formyl-methionyl-leucyl-phenylalanine. The interval between stimulations was 5 min. Intracellular free calcium concentration was monitored using the fluorescent calcium indicator FURA-2AM. Cytochalasin B (3.3 micrograms/ml) added 60 s before the cell stimulation enhanced all three polymorphonuclear leukocyte calcium responses by increasing the N-formyl-methionyl-leucyl-phenylalanine-induced calcium influx from the extracellular space. Cytochalasin B increased the peak intracellular free calcium concentration and elevated the plateau phase level, but had no influence on its shape. In addition, pretreatment with cytochalasin B of polymorphonuclear leukocytes suspended in low calcium medium restored their capacity to respond to a third stimulation with N-formyl-methionyl-leucyl-phenylalanine. Finally, in resting cells cytochalasin B caused a moderate increase in intracellular free calcium concentration which was independent of extracellular calcium.