The present report describes the increase in cytosolic-free calcium levels (Ca2+i) induced by chemoattractants in polymorphonuclear leukocytes (PMN) using the new fluorescent Ca2+ chelator Indo-1 AM. Increases in cytosolic Ca2+ were measured by flow cytometry. With this approach, 98% of PMN were found to respond to F-Met-Leu-Phe (FMLP) and leukotriene B4 (LTB4) stimulation. Although both substances induced a rapid and relatively homogeneous rise in Ca2+i, only FMLP gave a sustained Ca2+i rise, whereas that induced by LTB4 appeared transient. In addition, the combined use of wide-angle light scatter and electronic cell volume measurement allowed analysis of Ca2+i variations in the total peripheral blood cell population. The supernatant of a human fibrous histiocytoma cell line (GCT) was able to increase Ca2+ release in PMN. This activity may be ascribed to a new granulocytic activation factor, as neither human recombinant interleukin-1 alpha (IL-1 alpha), granulocyte-colony stimulating factor (G-CSF), nor granulocyte-macrophage-colony stimulating factor (GM-CSF), which were present in this supernatant, were able to induce a Ca2+i rise in PMN.