Aerolysin is a pore-forming toxin that plays a key role in the pathogenesis of Aeromonas hydrophila infections. In this study, we have analyzed the effect of aerolysin on human granulocytes (HL-60 cells). Proaerolysin could bind to these cells, was processed into active aerolysin, and led to membrane depolarization, indicating that granulocytes are potential targets for this toxin. Fura-2 measurements were used to analyze the effect of aerolysin on cytosolic [Ca
2؉] homeostasis. As expected for a pore-forming toxin, aerolysin addition led to Ca 2؉ influx across the plasma membrane. In addition, the toxin triggered Ca 2؉ release from agonist and thapsigargin-sensitive intracellular Ca 2؉ stores. This Ca 2؉ release was independent of the aerolysin-induced Ca 2؉ influx and occurred in two kinetically distinct phases: an initial rapid and transient phase and a second, more sustained, phase. The first, but not the second phase was sensitive to pertussis toxin. Activation of pertussis toxin-sensitive G-proteins appeared to be a consequence of pore formation, rather than receptor activation through aerolysinbinding, as it: (i) was not observed with a binding competent, insertion-incompetent aerolysin mutant, (ii) had a marked lag time, and (iii) was also observed in response to other bacterial pore-forming toxins (staphylococcal ␣-toxin, streptolysin O) which are thought to bind to different receptors. G-protein activation through pore-forming toxins stimulated cellular functions, as evidenced by pertussis toxin-sensitive chemotaxis. Our results demonstrate that granulocytes are potential target cells for aerolysin and that in these cells, Ca 2؉ signaling in response to a pore-forming toxin involves G-proteindependent cell activation and Ca 2؉ release from intracellular stores.